link.springer.com978-0-387-45524-2/1.pdfINTRODUCTION As is clear from a number of the chapters in...

INTRODUCTION

As is clear from a number of the chapters in this volume, 2-photonmicroscopy offers many advantages, especially for living-cellstudies of thick specimens such as brain slices and embryos.However, these advantages must be balanced against the fact thatcommercial multiphoton instrumentation is much more costly thanthe equipment used for confocal or widefield/deconvolution. Giventhese two facts, it is not surprising that, to an extent much greaterthan is true of confocal, many researchers have decided to add afemtosecond (fs) pulsed near-IR laser to a scanner and a micro-scope to make their own system (Soeller and Cannell, 1996; Tsaiet al., 2002; Potter, 2005). Even those who purchase a commercialmultiphoton system find that it helps to understand a bit more abouthow to optimize the performance of the fs laser system.1 ThisAppendix has been added to the Handbook to provide the basicalignment and operating information that such people need.

First, the safety announcement . . .

LASER SAFETY

Light sources for multiphoton microscopy are almost withoutexception very powerful pulsed lasers (laser class IV). It is vitalthat any personnel who perform alignment or other operations thatcarry a risk of beam exposure are familiar with and follow lasersafety regulations. During routine operation one MUST ensurethat accidental exposure to the pulsed laser beam is prevented byproviding proper shielding and interlocks.During alignment, protective eyewear is not an option — it isessential!See http://www.osha.gov/SLTC/laserhazards/ for US guidelines.

LASER ALIGNMENT

Just as in any other type of microscopy, correct optical alignmentis crucial for achieving optimal, diffraction-limited performance in2-photon microscopy. The alignment of external lasers such as theTi :S or similar 2-photon sources into a laser scanning microscopecan be simplified if a well-aligned “internal” or reference laser isavailable. In commercial confocal microscopes, typical candidatelasers include Argon-ion or green HeNe lasers or, more recently,

blue and green diode lasers. To provide an alignment beam towhich the external laser can be aligned, light from this referencelaser needs to be bounced back through the microscope opticaltrain and out through the external coupling port:CAUTION: Before you switch on the reference laser in thisconfiguration make sure that all PMTs are protected and/orturned off.

Place a front-surface mirror on the stage of the microscope andfocus onto the reflective surface using an air objective for conve-nience (at sharp focus, you should be able to see scratches or othermirror defects through the eyepieces). The idea of this method isto cause the reference laser beam to bounce back through theoptical train and emerge from the other laser port. To do this, selectfilter settings that will allow some of the light from the internallaser to exit the chosen coupling port. In order to bring two laserbeams to co-linearity, a beam-steering device is essential. A single-mirror beam steerer provides angular control while changing theseparation between the mirrors of a 2-mirror steerer provides beamtranslation (Fig. A1.1).

It is also possible to achieve beam translation with a secondangular control mirror. After adjusting the incoming near-IR beamto an intensity where it can be viewed without totally overwhelm-ing the reference beam,2 adjust one mirror to make both laser spotsmerge at the surface of the other (angle-adjustable) mirror. Thenthat mirror is adjusted to bring the beams to co-linearity. We findit useful to use a piece of light-blue paper as this shows the dimmedinfrared beam well. If the laser has been tuned to the far part ofthe spectrum, you may have to use an IR viewer or viewer card tovisualize the beam.

TESTING ALIGNMENT AND SYSTEM PERFORMANCE

On a regular basis and particularly subsequent to laser alignment,the performance of the multiphoton microscope should be tested.The prime indicator of proper alignment of an imaging system isits point-spread function, as measured by using a sample contain-ing sub-resolution fluorescent beads. A test slide can be preparedby letting a drop of diluted beads dry onto a coverslip. The beadsare then embedded in a drop of Sylgard elastomer (Dow Corning,USA) with a microscope slide placed on top. We usually use 0.2mm beads from Molecular Probes (Eugene, OR). These areavailable in a range of colors suitable for 2-photon microscopy. It

Appendix 1

Practical Tips for Two-Photon Microscopy

Mark B. Cannell, Angus McMorland, and Christian Soeller

900 Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006.

Mark B. Cannell, Angus McMorland, and Christian Soeller • Department of Physiology, FMHS, University of Auckland, New Zealand

1 The Multiphoton Users Group e-mail network at ·[email protected]Ò, operated by Steve Potter at Georgia Tech, enrolled its500th member in 2003.

2 As is explained below, this can be achieved by over-closing the slit and/orreducing pump power, because mode-locking is not required. We typicallyuse <20mw @ 800nm and <10mW at 720nm.

takes only about 30 minutes to prepare 10 slides in this way. Oncethe elastomer has set, these slides will last for months if kept in adark drawer. As a result, they provide a good standard to check themicroscope sensitivity and resolution provided you have recordedmicroscope and laser settings (including center wavelength, laserpower and bandwidth/pulse length) with each reference image.

With proper alignment, the beads should blur approximatelyevenly as you focus above and below them. Asymmetric blurringabove-and-below best focus indicates spherical aberration whilemotion of the centroid of intensity means that the objective aperture is filled asymmetrically. The spatial resolution (without apinhole) should be similar to confocal performance, valuesbetween 0.2–0.4mm in plane, full-width at half maximum(FWHM) and 0.5–0.8 mm out of plane (in the z direction) shouldbe attainable when using a high-numerical-aperture (NA ~1.3)objective.

A very weak and noisy signal can have a number of causes. Ifthere is no problem with the detectors or emission filters (most ofwhich would also be apparent when operating the microscope withconventional [1-photon] laser excitation), check that the laserbeam fills the objective rear aperture fully and evenly by rotatingthe objective turret to an empty position, placing a lens tissue overthe opening and inspecting the pattern of illumination (using an IRviewer if necessary). The beam should be accurately centered inthe empty socket and should form a uniform circle of light thatwill cover the rear aperture (~8–10mm wide) of a typical objec-tive lens. If the light intensity at the rear aperture is low (<10mW)make sure that no IR-opaque optical items are obstructing the illu-mination path.3 It is also possible that the beam is so badly mis-aligned that only scattered light is being observed. You can checkfor this by ensuring that adjustments of the alignment mirrors havethe expected effects on the spot in the BFP.

If the microscope is a combined confocal/multiphoton system,the bead slide is also a useful tool to disclose alignment offsetsbetween the 2-photon laser system and any other lasers. In partic-ular you should check for any axial offsets (i.e., focus shifts), par-

ticularly if the system is to be used for 2-photon flash photolysisor combined confocal and multiphoton co-localization studies.

In our laboratory we perform a basic system test with a pre-pared bead sample on a daily basis. This check (usually conductedfollowing system startup) is well worth the ~5 minutes it takes,especially if it helps avoid debugging signal problems later whena precious biological sample is on the stage.

LASER SETTINGS AND OPERATION

Historically, the mode-locked lasers used for 2-photon imagingcould be quite temperamental and ensuring that proper laser oper-ation was a large part of the challenge of running a multiphotonmicroscope. With the advent of fully computer-controlled turn-keylaser systems, this has become less of an issue. In any case, as themost versatile source for 2-photon imaging is still the tunable Ti :S laser in the femtosecond configuration, we will focus on ithere. Regardless of whether you are using a fully automated or amanually adjusted Ti :S system, it is important to monitor and optimize the laser output before imaging.

The choice of center wavelength is generally determined bythe fluorochromes to be excited. As a general rule of thumb youshould try to use the longest wavelength compatible with the dyesin your sample as this will help minimize photodamage and alsoreduce scattering of the excitation light. Data on excitation spectrais now available from many sources in the literature and, if indoubt, there are mailing lists where one can ask other researchersfor advice (see http://groups.yahoo.com/group/mplsm-users/ andhttp://listserv.acsu.buffalo.edu/archives/confocal.html).

MONITORING LASER PERFORMANCE

During tuning and imaging, laser operation can be very conve-niently monitored using a spectrum analyzer. We use a systemmade by Rees Instruments (currently available models include theRees E200 series laser spectrum analyzers by Imaging and SensingTechnology Ltd., Alton, UK) to monitor a secondary beam con-taining only a small fraction of the total output power. During lasertuning, this device allows one to measure the center wavelengthand, more importantly, the width of the spectrum. The spectral

Practical Tips for Two-Photon Microscopy • Appendix 1 901

A B C

FIGURE A1.1. (A) 2D simplification of the beam alignment process using a conventional beam-steerer. A vertical translation of a tilted mirror is used to bringthe two beams to a common point on a second, tiltable mirror. (B) Rotation of the second mirror at the point of the common spot makes the two beams co-linear. (C) The co-linear beams after alignment.

3 If little light is coming out of the objective, it may be the anti-reflection coat-ings that are at fault. Coatings used to reduce reflection losses in the visiblemay become mirrors in the near-IR. See the transmission tables in Chapter 7and its Appendix.

width of the beam, as displayed by the analyzer, provides the feed-back for optimizing the slit width and position to obtain mode-locked operation (with manually tuned laser systems). The start ofmode-locked operation is indicated by the change of the spectralshape from one or a small number of sharply defined lines whichindicate continuous wave (CW) operation, see Figure A1.2A, to an

approximately Gaussian-shaped output spectrum which may havea spike (Fig. A1.2B) indicating CW breakthrough. Optimal closureof the slit leads to a smooth Gaussian-like spectrum (Fig. A1.2C)which, in this case, is ~5nm wide (FWHM). At 750nm this spec-tral width implies a 120 fs pulse. Closing the slit further can leadto an oscillation of pulse amplitude (Q-switching), which is shown

902 Appendix 1 • M.B. Cannell et al.

A B

C D

E

G

F

FIGURE A1.2. (A–F) show spectrum analyzer outputduring Ti :S tuning. The small gradations at the bottomindicate 1nm. (A) Before mode-locking, the spectrumconsists of a few narrow spikes. (B) With mode-lockingunderway, the spectrum increases in width, but the spikeindicates CW breakthrough. To cure this, the slit needsto be closed more. (C) Optimal operation, the slit hasbeen closed just enough to stop CW but at the same timenot so closed that Q-switching starts, a mode of behav-ior shown in (D,E). To stop Q switching, more prismmust be inserted into the beam path (which will increasesystem bandwidth) and/or the slit needs to be opened (or even a reduction in pump power). (F) shows the short-est pulse that can be readily achieved with our CoherentMIRA 900F system. The FWHM of the spectrum is ~14nm at a center wavelength of ~750nm. (G) shows therelationship between the FWHM of the spectrum and thepulse width for a transform-limited sec2 pulse, withcenter wavelength indicated next to each curve. In thiscase, a 14nm bandwidth from our laser (F) implies a very short pulse width of 40 fs. Generally, we use longer pulses (~120fs e.g., C) than this in imaging experiments.

in the spectrum as oscillations (Figs. A1.2D, A1.2E) and should beremoved by re-opening the slit or increasing the intra-cavity groupvelocity dispersion by moving the intra-cavity prism further in. Bysuitable adjustment of the slit and the intra-cavity group velocitydispersion, the pulse may be shortened and this will be reflectedin an increase the width of the output spectrum (Fig. A1.2F). With our laser, a 14nm FWHM bandwidth can be achieved corresponding to a ~40fs pulse at 750nm. During imaging, Q-switching manifests itself as a sudden increase in image noise dueto aliasing between laser excitation and the pixel clock. A quicklook at the spectrum should indicate if the laser needs tuning toremove this source of image noise.

Typically, pulses leaving a commercial Ti :S laser, as used for2-photon microscopy, are ~100fs long. Pulse length is an impor-tant variable that is most accurately determined with an opticalautocorrelator. However, from a practical point of view, a spec-trum analyzer is easier to use than an autocorrelator and gives suf-ficient information on laser performance. The length of the laserpulse is inversely proportional to the spectral FWHM duringmode-locked operation. Figure A1.2G shows this relationship forvarious center wavelengths.

POWER LEVELS AND TROUBLE-SHOOTING

In our experience illumination power levels at the sample shouldbe kept <20mW in living cells to minimize the risk of cell damage,although that figure is dependent on the nature of the experiment,the 2-photon absorber, the objective NA, and sample scattering.

Problems with mode-locked lasers in 3D microscopic imagingmost often arise from:

1. Pump laser noise (amplitude noise or beam-pointing instability).

2. Pump laser alignment.3. Dirt on mirrors.4. Poor alignment within the cavity.5. Stray reflections from surfaces that reflect energy back into

the cavity.6. Poorly trained personnel changing the alignment between the

pump and the prisms of the Ti :S cavity over time.7. UFM (unidentified fingerprints on mirrors!).8. Air currents that affect beam-pointing stability.9. Loss of alignment of laser to microscope.

10. Poor matching of laser beam profile to microscope aperture.

To address problems 1–4, the manufacturer generally providestroubleshooting advice that should be consulted. Problem 5 can beavoided by using an optical isolator, i.e., a device which allowslight to pass only in the forward direction but blocks back reflec-tions. A simpler workaround (that has worked well in our hands)involves slightly tilting strongly reflecting surfaces (e.g., neutraldensity filters — see below) with respect to the optical axis. Forlaser safety you should provide an appropriate beam dump for anystrong reflections off the optical axis. Problems 6 and 7 should beresolved by the system manager. Problem 8 can be reduced by sur-rounding all beams with plastic tubes. Problem 9 can be ascer-tained using a reference laser, especially a laser built into themicroscope itself. Problem 10 arises from the laser beam being toosmall to fully fill the objective rear aperture (so a loss of resolu-tion occurs) or too large, in which case there is a loss of intensityat the sample. In both cases, the problem can be fixed using laserbeam expansion (or compression) with a telescope (Galilean beamexpander). In our microscope we use ~4x expansion of the Ti :S

laser beam as a reasonable compromise between filling the realaperture adequately and throughput. (We built a simple expanderfrom a plano-convex and a plano-concave lens which were single-layer antireflection coated.) In addition, by focusing the beamexpander carefully, it is possible to minimize the axial shift of focalplane between visible light and the IR.

CHOICE OF PULSE LENGTH

The dispersion of the pulse by the microscope optics is typically>2000fs2 at 800nm. This suggests that the shortest pulse width thatcan be delivered to the sample would be >100fs unless groupvelocity dispersion compensation is performed to “prechirp” thepulse (Soeller and Cannell, 1996). Shorter pulses increase the ratioof 3- to 2-photon excitation and, since 3-photon excitation at 800nm would correspond to hard UV, such excitation is generallyundesirable. We therefore suggest that for routine operation ~120fs pulses are probably optimal. Perhaps paradoxically, in theabsence of GVD compensation, a shorter pulse at the laser is trans-lated to a much longer pulse at the sample. As it is hard to run aconventional Ti :S laser with pulses longer than ~150fs, longerpulses at the sample may be produced by making very short pulses(e.g., 40 fs) at the laser. See Chapters 5 and 28 for further discus-sion on pulse broadening.

CONTROLLING LASER POWER

Being able to control laser power electronically is useful becauseit permits rapid suppression of the beam at the end of each scanline where the beam slows and stops before retracing its path. Thisslow movement subjects the parts of the specimen at either side ofthe raster to very high integrated excitation which is very damag-ing. Unfortunately, the acousto-optic modulators (AOM), whichare commonly used for this purpose in visible light microscopes,are less suitable for 2-photon because heating and birefringenteffects in the crystal reduce beam intensity stability. The square-law dependence of 2-photon excitation on input power amplifiesthis instability at the sample. Additionally, because the mode-locked laser beam has significant bandwidth (compared to a CWlaser) the beam will be dispersed if it is diffracted in the AOM. Asthe rear aperture of the objective must be overfilled, this disper-sion results in a loss of bandwidth and therefore a longer and mis-shapen pulse. This effect can be avoided if one uses the zero-order(i.e., undiffracted) beam of the AOM for microscopy and the firstorder beam is used simply to extract energy from it. However, asonly about 75% of the beam can be diffracted out, this approachonly reduces the beam to 25% of the input power.

A better alternative is to use a Pockels cell. While more expen-sive, these devices are much faster and more controllable than anAOM, but they also suffer from some problems:

1. The Pockels cell has a limited lifetime that is dependent on thetime spent in the energized state.

2. Alignment is critical: the full power of the beam must passcleanly through the free aperture and not touch the interior ofthe cell under any circumstance or damage will result.

3. High voltages are present.

It should be noted that, for ~120fs pulses, dispersive broadeningby the Pockels is generally small and should therefore be of noconcern when it is used in a 2-photon imaging setup.

If rapid beam modulation is not needed, laser power can becontrolled by neutral density filters or a polarizer. Such neutral


density filters need to be of the reflecting type as high powersdestroy absorbing filters. The beam reflected from the filter needsto be absorbed by something for safety and we use a “beam dump”made of black anodized aluminum with a machined recess so it ishard to see the dumped beam. Since the output beam of the laseris polarized, beam intensity may be modulated by rotating a polar-izer in front of it. Glass Glan-Thompson polarizers can be used butplastic polarizers are quite unsuitable for typical power levels asthey melt (see also Attenuation of Laser beams in Chapter 5, thisvolume.)!

AM I SEEING TWO-PHOTON EXCITEDFLUORESCENCE OR . . .

Sometimes it is unclear if a detected signal is due to multiphoton-excited fluorescence or if it is due to optical bleed-through of the(much more intense) near-IR excitation light. Such bleed-throughcan occur, for example, if one uses filters with an unknownresponse in the near-IR region. A simple test to distinguish betweenthese possibilities can be made by taking a control image with themultiphoton laser source running in CW mode (at similar power).When using a mode-locked Ti :S laser with manually operated slitthis can easily be achieved by over-closing the slit until mode-locking is lost and then reopening the slit with the starter mecha-nism disabled. If the signal in question disappears when using CWillumination, it must be due to some sort of multiphoton excitation(2- or 3-photon fluorescence, or second- or third-harmonic gener-ation). However, this simple test does not replace the more complexillumination-power vs. signal-intensity measurements needed tofully characterize each of these high-order excitation processes.

STRAY LIGHT AND NON-DESCANNEDDETECTION

One of the attractions of 2-photon microscopy resides in theimproved penetration depth obtained when imaging in stronglyscattering biological samples such as brain slices (see Soeller andCannell, 1999). Central to this advantage is the need to collectemitted photons that are also scattered and so may not be focusedby the microscope optics and are therefore lost at intermediateapertures. This problem can be overcome by using a photomulti-plier tube that is mounted close to the sample (so that the emittedlight does not pass through the scanning system) to create a “non-descanned detector.” Such detectors are arranged so that anyphotons of the right color, regardless of where they originate, aredirected onto the photocathode. As a result, non-descanned detection is also far more likely to pick up stray light from themicroscope surroundings than conventional confocal optics. Forexample, in a normal laboratory, light from computer screens andequipment LEDs can cause a strong background signal even whenthe room lights are turned off. To shield your setup from this straylight, you may need to fabricate suitable shields around the samplefrom black material. Alternatively, you may shield the wholemicroscope from the surroundings by enclosing it in a completelylight tight box. This can be conveniently combined with electricalshielding by providing a Faraday cage around 3 sides and the topof the instrument and fully closing it during imaging by drawinga black curtain or blind across the fourth side. For safety reasons,this cage and any blinds or curtain should be made from fire-proofmaterials.

LASER POWER ADJUSTMENT FOR IMAGING AT DEPTH

Although 2-photon excitation penetrates deeper into scatteringsamples (such as brain), the loss of peak excitation power at thefocus caused by scattering and spherical aberration still leads to aloss of signal at depth. The solution to this problem is to alter theillumination power as a function of depth and this is where theintensity modulation provided by the Pockels cell may be used to advantage. There are alternative ways to achieve changes in illumination power but all assume that the maximum power available from the 2-photon laser is higher than is needed fornormal operation. Thus, a wheel of reflective neutral density (ND) filters may be placed in the beam path, providing intensity controlto quantized levels appropriate for different imaging depths. A second option is to use a continuously variable reflective neutral density filter, which allows more precise control over laserpower, but requires either manual rotation during imaging or amotorized filter wheel. We suggest that the ideal solution is to automatically attenuate the laser beam. using a Pockels cell supplied with a varying drive voltage controlled by the focus position.

In our experience, the laser power needs to increase (roughly)exponentially with depth (e.g., see Fig. A1.4 in Soeller andCannell, 1999) but the exponential factor is highly dependent onthe sample. Thus a control experiment may be needed where asimilar sample is labeled with fluorescent beads (~2mm in diame-ter). For brain slices, or other tissues which can be perfused, thiscan be achieved by injecting the beads into a blood vessel beforeslicing. By imaging the beads at different depths, the depth depen-dence of the excitation may be determined and used in subsequentexperiments. (Using beads will give more reliable results thansimply staining the entire specimen with a dye as this avoids prob-lems arising from non-uniform staining.) It is important to notethat not all the signal loss is due to reduced excitation as emittedlight is also lost by scattering and adsorption. Thus, even if com-plete compensation of signal loss with depth can be achieved byraising excitation power, it is better to err on the side of caution asdelivering too much power into the preparation at any depth maylead to other concerns — for example, heating and other higher-order effects (see Chapter 38, this volume). If 100mW were deliv-ered and (eventually) absorbed within 10mm3 of tissue, theaverage rate of rise of temperature would be 2.5oC/s. Although thispower is close to the maximum that may be achieved by typical2-photon microscopes, it is clearly in the range where heatingeffects could become a serious problem.

SIMULTANEOUS IMAGING OF MULTIPLE LABELS

Another advantage of 2-photon excitation is that the 2-photon exci-tation spectra of fluorochromes are wider than their 1-photon coun-terparts. Multiple labels may therefore be imaged simultaneouslyby using a single excitation wavelength and multiple detectors withappropriate optics to isolate each different emitted wavelength.This approach has several benefits: (1) Removal of offset problemscaused by non-confocality of different lasers; (2) reduction inimaging time (which may be important for imaging of live-cellprocesses); (3) reduction in the total amount of laser exposure tothe tissue and (4) avoidance of chromatic aberrations. Care mustbe taken to ensure that bleed-through from one channel to another

904 Appendix 1 • M.B. Cannell et al.

is minimized by the use of the optimal beam-splitters (see alsoChapter 3, this volume). If some bleed-through is unavoidable thenan accurate measurement of the amount of bleed-through can bemade by imaging, in all channels, control slides that contain theindividual fluorochromes. From these measurements, contribu-tions from bleed-through from one channel to another can be esti-mated and removed by subtraction during post-imaging analysis(so-called “spectral unmixing”).

MINIMIZE EXPOSURE DURING ORIENTATIONAND PARAMETER SETTING

In most applications, imaging parameters need to be establishedby trial and observation prior to the commencement of imageacquisition. Common examples are scanning across tissue lookingfor “that cover image” and then establishing the upper and lowerlimits of a volume of interest. While the use of 2-photon excita-tion prevents photobleaching above and below the focal plane, in-plane photobleaching can be severe and care must be taken toavoid over-exposure of samples to illumination light during theseadjustment procedures.

The key is to think before imaging. For example, if the sampleneeds to be located in focus, is full power really necessary or willthe detection of just a few photons be sufficient? It follows thatduring setup, the detector gain should always be set high and laserpower as low as possible. Single scans should be used in prefer-ence to continuous scanning. Can the sample be moved to an unex-posed region once the acquisition parameters are set? Once thecorrect settings have been determined, then laser power can beincreased for actual imaging and focal-plane bleaching indicatesthat the maximum amount of information available has beenextracted from the dye in the sample.

ULTRAVIOLET-EXCITED FLUOROCHROMES

The use of ultraviolet (UV) excited dyes in 1-photon imaging isrestricted by the opacity of conventional optical components at UVwavelengths as well as by chromatic aberrations and by the cost

and size of UV lasers. 2-photon excitation of UV dyes does notsuffer from these problems because the excitation wavelengths arenear-infrared, in a range that is compatible with normal optics. The ability to use UV dyes allows more labels, and colors, to beused in multiple-labeling experiments. In addition, combining aUV-excited probe emitting in the blue part of the spectrum allows greater spectral separation from a yellow-red label. UVdyes, in general, may be excited by 2-photons at wavelengths £750nm. For example, the AlexaFluor 350 fluorochromes (MolecularProbes, Eugene, OR) come in a range of forms. The near-UV-excited nucleic-acid probes DAPI and Hoechst are often sowell excited using 2-photon illumination that it is necessary to use very low concentrations to prevent bleed-through into otherchannels.

ACKNOWLEDGEMENTS

We would like to thank Tim Murphy (University of British Columbia, Vancouver, CA) for helpful comments on the manuscript.

REFERENCES

Potter, S.M., 2005, Two-photon microscopy for 4D imaging of living neurons.In: Imaging in Neuroscience and Development. A Laboratory Manual, (R.Yuste, and A. Konnerth, eds.), pp. 59–70, Cold Spring Harbor LaboratoryPress.

Soeller, C., and Cannell, M.B., 1996, Construction of a two-photon microscopeand optimisation of illumination pulse width. Pflugers. Archiv. 432:555–561.

Soeller, C., and Cannell, M.B., 1999, Two-photon microscopy: Imaging in scat-tering samples and three-dimensionally resolved flash photolysis. Microsc.Res. Tech. 47:182–195.

Taal, P.S., Nishimura, N., Yoder, E.J., White, A., Doluick, E., and Kleinfeld,D., 2002, Principles, design and construction of a two-photon scanningmicroscope for in vitro and in vivo studies, In: Method for in vivo OpticalImaging, (R. Frostig, ed), CRC Press, pp. 113–171.


INTRODUCTION

Since biologists became aware of the confocal microscope in thelate 1980s, numerous optical designs have been introduced bymanufacturers to try to meet the often-contradictory requirementsof the biological microscopist. Although many of these designs arediscussed at greater length in other chapters of the Handbook, itwas thought that it might be both useful to the reader, and fairerto those designs not discussed elsewhere, to provide the readerwith a concise compilation of all the designs now available.

To that end I requested optical diagrams and tabular informa-tion from all of the major suppliers of the instruments used by biol-ogists for 3D microscopy1 and the items that they provided makeup the bulk of this Appendix. Often manufacturers were hesitantto provide specific information about details such as PMTs or scan-ning speeds etc., because they realized that there was a good

chance that such data would go out of date with their next productannouncement. However, I tried to apply the same criteria to allthe contributors and this is as good a place as any to thank the man-ufacturers for their splendid cooperation.

To assist the reader, some of the optical information consid-ered most relevant to the optical performance of these instrumentshas been collected in Table A2.1. Although such a table cannotcontain all of the relevant information about such complex instru-ments, the headings have been chosen to reflect those specifica-tions indicated to be of prime importance in the other chapters ofthe Handbook. Abbreviations are explained in the footnote.

Of course, the manufacturers are correct about this informa-tion going out of date. Fortunately the WWW is now there to bringyou up to date. Even when the models are all different, we hopethat the you find the column headings in the table of optical param-eters useful as the basis of questions you might ask about futuremodels.

There has been no effort to compare the computer operatingsystems used to control these instruments. I wish to emphasize thatthis is not because I think such details unimportant, but ratherbecause software systems tend to change with great speed and, in addition, operating systems are probably best assessed in person.

Appendix 2

Light Paths of the Current Commercial Confocal LightMicroscopes Used in Biology

James B. Pawley


James B. Pawley • University of Wisconsin, Madison, Wisconsin, 53706

1 We have neglected to include any information on the systems for widefield/deconvolution only because the optical paths of such systems are fairlystraightforward, and not in need of explanation.

Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology • Appendix 2 907

FIGURE A2.1. Schematic of the BD-CARV IIlight path. The variable intensity light from aHg/metal halide light source passes through an exci-tation filter before being defleted by a dichroic mirrortowards the sample. The excitation light passesthrough a Nipkow spinning-disk containing multiplesets of spirally-arranged pinholes placed in the intermediate-image plane of the objective lens. Thecolumn of excitation light is projected through 1000pinholes to simultaneously scan the entire field onceevery millisecond, thereby creating a full image ofthe focal plane in real-time. The emitted light passesthrough the dichroic mirror and the emission filterbefore either entering the CCD camera or the binoc-ular eye-piece. The pinhole disk can be moved in andout of the light path to produce a confocal or a widefield fluorescence image. A variable slit at theimage plane can be used to selectively illuminate anarea of the sample allowing Fluorescence RecoveryAfter Photobleaching (FRAP) to be performed. Allmovable parts including the filter wheels, spinning-disk shutters, and mirrors are automated and are con-trolled via touchpad or third-party software. Figurekindly provided by BD-Biosciences, (Rockville,MS).

FIGURE A2.2. Schematic of the LaVision-BioTec TriM-Scope light path. Multifocal multiphoton microscopy using a beamsplitter built with flat optics. Lightfrom a fs, near-IR, pulsed laser first passes a polarizing attenuator and a beam-expander before entering a pre-chirp compensator. It is then formed into as manyas 64 beams of equal intensity and spacing by being reflected from an array of sliding, planar, optical elements. The linear array of beams is then deflected by2, closely-spaced galvanometer mirrors and fed into the microscope by being reflected off a high-pass beam-splitter. Two-photon-excited fluorescence from anydye located at the focus plane of the objective passes through the short-pass dichroic, and barrier filters to a CCD camera or other photodetector.2 Because ofthe large number of parallel beams and the high-QE of the CCD camera, it is possible to obtain useful, optical-section images at up to 3.5k frames/second and,because the system relies on 2-photon excitation, bleaching is restricted to the focal plane. For more discussion see Chapter 29, this volume.

2 T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, (2001), High efficiency beam splitter for multifocal multiphoton microscopy, J. Microsc., 201:368–376.

908 Appendix 2 • J.B. Pawley

Table A2.1. Optical Parameters of Current Commercial Confocal Microscopes

ScannedRetrace Fastest field, Largest

protect/Laser Fiber Beam line scan, Pixel diam. int RasterCompany Model Lasers/Arc atten optics Pre-optics expander Scanner Hz times im. plane Pixel

BD- CARV II X-Cite 120 NA/intensity Liquid- NA NA Single-sided 1 k fps ~1 ms 21 mm CCDBiosciences Hg/halide controlled by filled light Petran disk 5 k rpm

arc, 8-place aperture guidefilter wheel

Lavision- TriMScope2 Ti-sapph NA/ Laser is NA Yes 1–64 beams 3.5 k fps > 500 ns 20 mm, CCDBiotech 750–100 nm, Attenuator coupled scanned by 2 adapted to

100 fs pulse 0.1–100% directly galvos CCD used

Leica TCS SP2 AOBS Many, Yes/3 SM-PP Laser-merge Adjustable Rotatable k-scan 1.4 k or >500 ns 22 mm 4096 ¥ 4096351–633 nm AOTFs 2.8 k in

bi-direct

MP RS Ti-Sapph Yes, EOM SM-PP Laser-merge Adjustable Rotatable k-scan 4 k, or 8k >500 ns 22 mm 4096 ¥ 4096in bi-direct

Nikon C1-plus Up to 3, AOM (opt) SM-PP Laser-merge Fixed 2 close galvos 500, 1k >1.68us 17 mm 2048 ¥ 2048408–638 nm in bi- @512 ¥

direct 512

C1si Up to 3, AOM SM-PP Laser-merge Fixed 2 close galvos 500, 1k From 17 mm Up to408–638 nm laser input in bi- 4.08us w/scan 512 ¥

fiber. MM direct at rotation 512 inemission 512 ¥ 512 spectralfiber in mode

spectralmode

Olympus FV 300 Many, Yes, AOTF SM-PP Laser-merge Fixed 2 close galvos 1 k or 2 k >2 ms 20 mm 2048 ¥ 2048405–633 nm bi-directIR port

FV 1000 Many, Yes, AOTF SM-PP Laser-merge Fixed 2 pair of close 2 k or 4 k >2 ms 18 mm 4096 ¥ 4096active galvos, separate bi-directstabilizer, image/bleach351–633 nm, scanners,IR port circular bleach

DSU Hg arc NA/intensity NA NA NA Interchangeable 3 k rpm, >1 ms 18 mm CCDcontrolled by single-sided slit- 15 fpsaperture pattern disk,

3 k rpm

Visitech VT-infinity Many, Yes, AOTF SM-PP Laser-merge NA Single galvo 2 kHz 2 ms 17 CCD405–647 njm (opt) scans an array

of point sourcesVT-Eye Many, Yes, AOTF SM-PP Laser-merge Fixed 1 galvo, 1 AOD 50 kHz 20–125 ns 1024 ¥ 1024

351–647 nm (opt)

Yokogawa5 CSU 10 2 or 3 lines6 NA/AOTF SM Laser-merge Fixed Double Petran 1800 rpm, ~1 ms 13 ¥ 9.5 mm CCD3.5 mm Disk w/micro- 360 fpscore lenses

CSU 22 3 or 4 laser NA/AOTF SM Laser-merge Fixed Double Petran Variable, ~1 ms 13 ¥ 9.5 mm CCDlines 3.5 mm Disk w/micro- to 5 k

core lenses rpm, 1 kfps

Zeiss LSM510META Many, Yes, AOTF SM-PP Laser-merge Adjustable 2 close galvos 1.3 k or 640 ns– 18 mm 2048 ¥351–633 nm 0.05–100% 2.6 k in 2.3 ms 2048(Ti : Sapph) (AOM) bi-direct

LSM 5 Pascal Many, No/ SM-PP Laser-merge Adjustable 2 close galvos 1.3 k or 640 ns– 18 mm 2048 ¥ 2048405–633 nm Mechanical 2.6 k in 2.3 ms

attenuator bi-direct0.05–100%

LSM5-LIVE Many, Yes, AOTF SM-PP Laser-merge Adjustable 1 galvo (>60 k) 16 ms– 18 mm 1024 ¥ 1024405–635 nm 0.05–100% Cylindrical line-scan 120 fps, 20 ms

512 ¥ 512,1010 fps,512 ¥ 50

1 Record transmitted light through disk.2 As the TriMScope is actually a multi-focus multiphoton fluorescence illuminator with widefield detection onto a CCD, its performance depends a great deal on the per-formance of this device.3 These numbers assume that the tube mag is 1¥.


Table A2.1. (Continued)

Zoom Tube Beam Beam- Pinhole Spectral Reflected/range mag dump splitter alignment Pinhole range selection Photodetector Channels transmitted Digitizer z-motion

NA 1.1¥ NA 5-place Self- Fixed 70 mm, 8-place filter CCD or EM- 2-camera port No/yes1 CCD Piezodichroic aligning 180 m spacing wheel CCD ±100 nmwheel (opt)

ROI for 1¥ NA Short-pass NA 8-place filter CCD or EM- 3-camera port Yes/no CCD, StepperCCD mode dichroic (multiphoton wheel, CCD, 1–32 (opt ion, motor,

excited spectrometer PMT array PMT, (peizoonly) (opt) 12-bit) opt)

32 : 1 Yes Acousto- Preset Common Prism, 4 PMT, 8 Yes/yes 12-bit Galvo,Optic pinhole, adjust motorized cooling ±40 nm

20–800 mm mirrors option,APD option

32 : 1 Yes Acousto- Fixed Common Prism, 4 PMT, 8 Yes/yes 12-bit Galvo,optic pinhole, adjust motorized cooling ±40 nm

20–800 mm mirrors option, +2non-descanned

infinite 3.8 Yes Dichroic, Fixed Common Replaceable 3 side-window 4 Yes/yes 12-bit Stepper,changes w/focusable, pinhole, filter cubes fiber-coupled ±50 nmwith cube alignable 30, 60, 100, PMTs

pinhole lens 150 mm3

infinite Dichroic, Fixed Common 3 diffraction 32 element 32 acquired Yes/yes 12-bit Stepper,changes w/focusable, pinhole, gratings for multianode simultaneously 50 nmwith cube alignable 30, 60, 100, 2.5 nm, 5 nm, PMT increments

pinhole lens 150 mm4 and 10 nmchannel width

10 : 1 3.42¥ Yes Dichroic, Common 5 sizes Dichroic filter 3 PMTs, 2 3, 2 fl, 1 trans Yes/yes 12-bit Stepper,(infinity) cubes, 2 pinhole, cube fluor, 1 trans ±10 nm

positions alignable50 : 1 3.82¥ Yes Dichroic Common adjust 2 diff-grating 5 PMTs, 2 5, 4 fl, 1 trans Yes/yes 12-bit Stepper,(infinity) wheel, 6 pinhole, 50–800 or channels, spectral, 1 ±10 nm

positions alignable 50–300 on motorized trans, Photonspectral slits counting mode

NA 1¥ No Filter cube Self- Vert & horiz Dichroic filter CCD or 1 No/yes CCD Stepper,aligning slits, cube EM-CCD ±10 nm

5 sizes

NA No Dichroic, preset, rect. ~1 k fixed, Dichroics/ CCD CCD Yes/no CCD Piezo,4 positions array 50 mm filters ±100 nm

adjustable50 : 1 No Dichroic, preset/ 5 slits, Dichroics/ 4 hi-QE PMTs 4 Yes/yes 10 bits Piezo,

6 positions adjustable 10–100 mm filters ±100 nm

NA 1¥ NA 1 dichroic, Self- 50 mm, 20 k on Dichroics/ CCD or 2-camera port No/no* CCDexchangeable aligning disk, ~1 k Filters, EM-CCDby user /FOV 3 emisson,

3 barrierNA 1¥ NA Dichroic, Self- 50 mm, 20 k on Dichroics/ CCD or 2-camera port No/no* CCD

3 positions aligning disk, ~1 k Filters, EM-CCD/FOV 3 emisson,

3 barrier

0.7–40¥ 0.84¥ Yes Dichroic, 4 x,y,(z), 3, 200 steps, 3 dichroics, 3/4 filtered 8 Yes/yes 8–12-bit Microscope4 positions diameter 0.1–13 Airy 6 positions, + PMTs;&/or 10 nm,

adjustable Units 10–1 k spectral diff. Grating Piezo,mm detector, 10 nm w/32 mPMTs ±5 nm

/channel array,7

0.7–40¥ 0.84¥ Yes Dichroic, 2 x,y, 1, 200 steps, 2 dichroics, 2 filtered 4 No/yes 8–12-bit Microscope2 positions diameter 0.1–13 Airy 6 positions PMTs, 10 nm,

adjustable Units trans PMT Piezo,10–1 k mm ±5 nm

0.5–2¥ 1.18¥ Yes Achrogate, 2 17 slits, 0.5–10 detector 512 ¥ 1 linear 2 No/no 8–12-bit Microscopeline-mirror adjustable Airy units dichroic, CCD 10 nm,on clear 12 positions, Piezo, ±5 nmblank 8 position

barriers

4 These numbers assume that the tube mag is 1¥.5 Yokogawa scanners are manufactured by Yokogawa Electric (Tokyo, Japan), but retailed by a number of companies including, Andor Technologies (Belfast, UK), SolamereTechnology (Salt Lake City, UT), PerkinElmer (Downer Grove, Il), Visitech (Sunderland, UK).6 It is possible to use 4 lasers with a quad, dichroic beamsplitter.7 Transmission PMT and 4-channel non-descanned PMT detector also available.

1. Detection channels with stepless tunable bandpass and PMT2. Beam splitter or mirror for auxiliary emission outlet (optional)3. Emission filter and polarization filter (rotatable) (optional)4. Excitation pinholes (excpt. IR)5. Merge module. Combination of up to 4 visible lasers6. a) Multiline Ar-Laser (457 – 476 – 488 - 496 – 514) b) HeNe Laser 543

c) HeNe Laser 594 d) Kr Laser 568e) HeNe Laser 633 f) IR Laser TiS for

Multiphoton excitationg) HeCd Laser 442 h) Solid state Laser 430i) Ar Laser 351 – 364 j) Diode Laser 405

7. EOM for intensity control of IR Laser8. AOTF for intensity control on VIS and UV Lasers9. Variable adaptation optics for UV / 405nm illumination

10. K-Scanning module for optically correct scanning method and field rotation11. Scan lens12. Beam splitter for non-descanned reflected light mode (optional)13. Objective optics14. Sample15. Condensor optics16. Detectors for non-descanned transmitted light (optional)17. Secondary beam splitter for NDD transmitted light (optional)18. Secondary beam splitter for NDD reflected light (optional)19. Detectors for non-descanned reflected light (optional)20. Beam splitter for UV illumination (optional)21. Variable beam expander optics22. Beam splitter for IR or violet illumination (optional)23. Acousto Optical Beam Splitter (AOBS )24. Pinhole optics25. Detection pinhole26. Spectral detector prism

FIGURE A2.3. Schematic diagram of Leica TCS SP2 AOBS. The Leica TCS SP2 AOBS is anadvanced confocal microscope in which all filtering and beam-splitting functions are performedby either liquid-crystal or acousto-optical components. This makes the system extremely flexiblein terms of being able to add new lasers or adapt to new emission bands. The acousto-opticalbeam-splitter (AOBS) is essentially transparent except at exactly the laser wavelengths (see Fig.3.23). The K-scan galvanometer mirror arrangement is capable of being rotated around the opticalaxis to change scan directions. There is one adjustable pinhole for all 4 prism/moving-mirror spec-tral-detection channels. Leica also makes the TCS SP5, which is similar but employes a tandemscan system which permits one to switch between a scanner employing a normal, analog gal-vanometer and one employing resonant galvanometer for high-speed, bi-directional scanning atup to 16k lines/s.

1. TiS Laser (pulsed IR)2. EOM for intensity control of IR Laser3. K-Scanning Module for optically correct scanning

method and field rotation4. Scan optics5. Beam splitter for non-descanned reflected light mode

(optional)6. Objective lens7. Sample8. Condensor optics9. Detectors for non-descanned transmitted light

10. Secondary beam splitter for NDD transmitted light11. Secondary beam splitter for NDD reflected light

(optional)12. Detectors for non-descanned reflected light (optional)13. Variable Beam expander optics

FIGURE A2.4. Schematic of the Leica MP RS Multiphoton Fluorescence Microscope. The LeicaMP RS is a single-beam scanning fluorescence microscope that uses a ps near-IR laser light source toproduce optical-section images of suitable specimens. It is designed for viewing living cells and incor-porates a variety of non-descanned detectors to record both transmitted and backscattered fluorescencesignal. This instrument uses a fs-pulsed, near-IR laser multiphoton excitation and a high speed gal-vanometer to provide fast imaging. Figures kindly provided by Leica Inc. (Heidelberg, Germany).

PMT 2

PMT 3

PMT 1

EmissionDichroic 1

SMA Connector

Nikon EF-4Filter Block

Nikon EF-4Filter Block

CH2Emission

Filter

CH3Emission

Filter

CH1Emission

Filter

Galvanometerfrom Laser

ModulePair

PrimaryDichroicMirror Mirror

FixedMirror

PinholeLens

Prism

Scan Lens

MountingAdapter

to Microscope

PinholeTurret

Mirror

Lens

EmissionDichroic 2

Multi-anode PMT

Multiple Gratings(2.5/5/10nm)

SPECTRALDETECTOR

SCAN HEAD

3-COLORDETECTOR

Unpolarized Light

OpticalFiber

PolarizationRotator

B

A

P

Polarized BeamSplitter

FIGURE A2.5. Schematic of the Nikon C1si light path. The C1-Plus is a 3-channel fluorescence plus transmission, single-beam, galvanometer-scanned, con-focal microscope. Because both the lasers and the PMTs are located externally and coupled through fibers, the C1 scan head is extremely compact and is veryeasy to move from one microscope to another. The standard unit includes laser module, in which a wide variety of gas and solid-state lasers can be installed. ascan-head and a DU-3 three-PMT detector module containing the collimating and focusing lenses, and photomultiplier tubes. The “si” version includes an addi-tional sophisticated spectral detector that is also coupled to the scan head through a multi-mode fiber. The detector itself incorporates a Diffraction EfficiencyEnhancement System (DEES) in which a polarized beam splitter separates the unpolarized signal beam into two parts (red and blue lines). One part passesthrough a prism polarization rotator so that all the light strikes the diffraction grating with the optimal (s-plane) polarization to be diffracted with maximum effi-ciency by one of 3 gratings (2.5, 5 and 10nm/channel). Both ray bundles are then focused onto a 32 channel micro PMT by a pair of reflecting lenses (A andB). Simultaneous readout is possible from all channels. The digitization system uses 2 sample-and-hold circuits to optimize signal integration. Figures kindlyprovided by Nikon Inc. (Tokyo, Japan).


gnitarG

gnitarG

tilS

tilS

2TMP

1TMP

3TMP

4TMP

resaL1 trop

resaL2 trop

resaL3 trop

jbo oT ce evit snel

gninnacSsrorrim

lif noissimE t sre

ticxE a noitsrettilpsmaeb

lacofnoCelohnip

aeb noissimE m ilps tt sre

morf resaL lanoitpo rennacs

el lacofnoC sn

A

fsnart lipuP snel re

snel ebuT

FIGURE A2.6. (A) Schematic of the Olympus Fluoview 1000. The Fluoview 1000 is the most recent single-beam laser-scanning confocal fluorescence micro-scope introduced by Olympus. It offers 4 separate fluorescence detection channels, two of which incorporate diffraction gratings and adjustable slits to tune thepassband. Besides the normal scanning mirrors there is a second independent SIM-scanning arrangement (not shown in the figure) to control lasers used forphoto-uncaging or for intentionally bleaching the specimen. To keep the signal up when the light dose to the specimen must be kept low, this new scanner notonly incorporates dichroic elements employing “hard’ coatings to ensure the highest transmission, it also offers a photon-counting option to reduce PMT multi-plicative noise. Figures kindly provided by Olympus Corp. (Tokyo, Japan).


LW

C

DSU

DSUFluorescentmirror unit

Disk box

Lightilluminator

Monitor

CCD camera

Imaging lens

Camera adapter

ND filterLightsource

Rotary disk

IlluminationtubeImaging lens

Objective lens

Specimen

B

FIGURE A2.6. (Continued) (B, C) Schematic of the Olympus DSU disk-scanner. The Olympus DSU is a disk-scanning confocal fluorescence microscopethat uses a mercury arc for excitation. The optical system is identical to that used for normal epi-fluorescence with the exception that an opaque disk is locatedin the intermediate image plane. Slits in this coating on this disk allow light to reach the focus plane and prevents light from this plane from reaching the CCDcamera. To keep the light dose to the specimen low, this new scanner not only incorporates “hard” coatings to ensure the highest transmission of the dichroicelements, it also offers a photon-counting option to reduce PMT multiplicative noise. (C) Layout of one of several interchangeable scanning disks used in theOlympus DSU disk-scanner. The thickness and spacing of the slits varies on the 5 available disks have each been optimized for use with a particular objective.Figures kindly provided by Olympus Corp. (Tokyo, Japan).

Micro lens array

Dichroic

Galvo scanner

Pinholes

Imageplane

A

Primarywheel

Barrierwheel

Slit

PMTlens(e)

AOD(astigmatism)

Fiber

Negativecylinder

Positivecylinder

AOD input

Scan

Field

B

Collimating

FIGURE A2.7B. Schematic diagram of Visitech VT-eye. The VT-eye incor-porates a novel acousto-optical deflector (AOD) scanner, that combines ultra-fast horizontal scanning to provide high-resolution confocal imaging forreal-time, living-cell confocal microscopy. The AOD scans the X axis at up to50,000 lines/s or 400 frames/s, fast enough to capture clear images of dynamicevents such as Ca++ puffs, sparks and waves. Multi-wavelength imaging formulti-labeled specimens from UV through the visible to the near infrared isachieved by using a selection of motorized, primary multi-band dichroics. Thesystem operates with almost any laser, or combination of lasers, and uses AOTFtechnology to provide fast laser-line selection. The VTeye comes with up to 4high-QE PMTs. The piezoelectric focusing system is capable of changing focuspositions at up to 100 slices per second. Although high-speed acquisitioncreates vast quantities of data in a very short time, hours of experiments maybe recorded at the maximum capture rates on a range of parallel, hard-diskmodules. Figure kindly provided by Visitech Inc. (Sunderland, UK).

FIGURE A2.7A. Schematic diagram of Visitech VT Infinity. The optical path starts with a stationary micro-lens array illuminated by an expanded laser beam.A galvanometer mirror (x) incorporating a piezoelectric micro-deflector (y) scans the array to cover the sample and then de-scans the returning fluorescencesignal. This light is separated from the illuminating beam by a dichroic mirror, and passes through a stationary pinhole array to create confocal data. This datais re-scanned, in perfect synchronization, by being reflected off the reverse side of the galvanometer mirror onto a sensitive CCD camera. The galvanometerscanner is readily synchronized to the camera capture parameters, both exposure time and frame capture rate. Either multiple-line lasers or multiple lasers in anycombination can be coupled through an AOTF that provides high speed (~ms) laser-line selection and intensity control. Laser excitation can be coupled in eitherby optical fiber or by direct coupling. Motor-driven filters change dichroic and detection bandpass. This system couples the advantages of high-brightness, laserillumination with multipoint scanning to keep the instantaneous intensity down while providing a data rate high enough for fast image detection, using a high-quantum-efficiency CCD camera. Figure kindly provided by Visitech Inc. (Sunderland, UK).


Laser for excitation

Microscope

Eyepiece

Dichroic mirror(triple)

Barrier filter(triple)

Exciter filter(triple)

Microlens array disk

Pinhole array disk

Port select mirror

Optical Path of CSU22

Camera port

ND filter & Shutter

excitation laser

fluorescence A

B

Fiber Input

CameraDetector

Microlens Disk

Relay Lens

Filter Wheel

Relay Lens

Pinhole Disk

MicroscopePort

CollimatingLens

FIGURE A2.8. Schematic of the Yokogawa CSU 22. The Yokogawa scanner was the first disk scanner to offer both laser illumination and multibeam excita-tion. The mircolenses increase the efficiency of the illumination path from the 2–10% common to ordinary disks to almost 60%. (A) Laser light enters the scanhead through a single-mode optical fiber, reflects off a mirror and through one of 3 exciter filters. After passing through an ND filter, and a beam expander, itilluminates the microlens array on the top disk of the rotating scanning assembly. The lenses focus the light through a short-pass dichroic and onto the array ofpinholes in the lower disk. As this disk is in an image plane, the light passing each pinhole is focused into a point at the focus plane of the objective. Fluores-cent light returning from the focus plane passes up through the pinholes, and reflects off one of 3 dichroic mirrors located between the two disks and into thedetection path. After passing through one of 3 barrier filters, a selection mirror sends this light either to the camera port or to the eyepiece. (B) Simplified rayoptical diagram of the CSU-22. The pinhole disk resides in an image plane and the signal passing the pinholes is first made parallel by a relay lens, then passedthrough the emission filter before being focused onto the CCD chip by a second relay lens. Other details shown in Figure 10.9. Figures kindly provided byPerkinElmer Corp. (Shelton, CT).

A

FIGURE A2.9A. Schematic diagram of ZeissLSM-5-LIVE Fast Slit Scanner. The LSM-5-Live isa line-scanning confocal microscope using line illu-mination and a linear detector. Because it illuminatesabout 100x more points than does a single-beaminstrument, the LSM-5 Live can acquire data at amuch higher speed while still keeping the peak lightintensity low enough to avoid singlet-state saturation.In addition, the quantum efficiency of the linear CCDis about 10x greater than that of most PMTs. Laserlight enters the scan head through optical fibers (1)where it is combined by a series of mirrors (2, 3) andthen passes to beam shaper (an expander and a cylin-drical lens that converts the collimated Gaussianbeam into laser light with a rectangular cross-section)(4) and also focuses it precisely onto the AchroGatebeam splitter (5), reflects all wavelengths but onlyalong a reflective line across its center. As a result, nomatter what the wavelength, it reflects 100% of thelaser light but passes >95% of the signal light to thedetectors. The size of the raster on the specimen iscontrolled by a 0.5–2x zoom optic (6), that feeds thelight to the y-scanning mirror (7), through the scanlens (8), the objective lens (9) and on to the speci-men, (10). Returning signal follows the same path butmostly misses the reflective strip in the Achrogate andproceeds through a wheel of secondary dichroicbeam-splitters (11) to one of 2 tube-lenses (12) thateach focuses the line illuminated in the specimen ontoa 17-position, slit aperture plate (13). Light passingthe slits is first filtered by emission filters (14) andthen detected by a 1 ¥ 512 linear CCD detector (15)(see also Fig. 9.6).

B

PMTA

G

PMT

PMT

L

L

CL BC

M

O

S

NDD

MDBS

SCXY

DBS

DBS

EF

EF

PH

PH

PH

M Spectral Imaging

Imaging

Excitation

FO/EPD

LCLMBCMDBS

SCXYOSPH

DBSEFPMTGPMTA

NDDFOEPD

lasercollimator lensmirrorbeam combinermain dichroic beam splitter

scanner X/Yobjectivesamplevariable pinhole

dichroic beam splitteremission filterphoto multiplier tubegratingPMT array (META)

non-descanned detectorfiber outexternal photodetector

FIGURE A2.9B. Optical beam path of the Zeiss LSM 510 META. A unique scanning module is the core of the LSM 510 META. It contains motorizeddichroic mirrors and barrier filters, adjustable collimators, individually adjustable and alignable pinholes for each of 3 (or even 4) detection channels, as well asscanning mirrors, and highly sensitive PMT detectors including the 32 micro-PMTs of the META spectral detector. All these components are arranged to ensureoptimum specimen illumination and efficient collection of reflected or emitted light. The highly optimized optical diffraction grating in the META detector pro-vides an innovative way of separating the fluorescence emission spectrum to strike 32 separate, micro-PMTs, each of which covers a bandwidth of ~10 nm.Thus, a spectral signature is acquired at each pixel of the scanned image. Such a dataset can subsequently be digitally “unmixed” to separate signals from dyeswith overlapping emission spectra. The Beam Path: (1) Optical Fibers, (2) Motorized collimators, (3) Beam combiner, (4) Main dichroic beamsplitter, (5) Scan-ning mirrors, (6) Scanning lens, (7) Objective lens, (8) Specimen, (9) Secondary dichroic beamsplitter, (10) Confocal pinhole, (11) Emission filters, (12) Pho-tomultiplier, (13) META detector, (14) Neutral density filter, (15) Monitor diode, (16) Fiber out.


C

HAL

HBO

Mirror

Collimator

VIS Fiber

UV Fiber

LSFMonitor Diode

NDF

PinholeOptica

MDBS

DBS2DBS1DBS3

PMT3

VP3EF3

VP2EF2

VP4EF4

VP1EF1

PM

T2

PMT4

λ-selectiveElement

SpectralDetector

FiberCoupler

AOTFShutter

FiberCoupler

AOTFShutter

Ar-

UV

Las

er o

r41

3 nm

Ar/

ArK

r La

ser

HeN

e La

ser

HeN

e La

ser

PM

T1

xy

DBC

Plate

ScannerScanLens

Eyepiece

Tube Lens

T-PMT

CondensorSpecimen

Objective

Tube Lens

Collimator

Pinhole Optics

MDBS

TV

DBS1EF1VP1 EF2

APD1

APD2

VP2

Inverted Microscope

Scan Module on Side Port

APD UnitFCS on Base Port

LaserModule UV

LaserModule VIS

FIGURE A2.9C. (Continued) Schematic diagram of Zeiss LSM FCS showing how the fluorescence-correlation spectroscopy (FCS) unit is attached via thebase port of the Axiovert 200M microscope while the LSM 510 META is attached to the side camera port. All figures kindly provided by Carl Zeiss Inc. (Jena,Germany).

INTRODUCTION

The electronic structure of crystalline Si is such that electromag-netic waves having the energy of light photons (1.75–3.0 electronvolts) can be absorbed to produce one free or “conduction” elec-tron. If an image is focused onto a Si surface, the number of thephotoelectrons (PE) produced at each location over the surface isproportional to the local light intensity. Clearly, all that is neededto create an image sensor is a method for rapidly converting thelocal PE concentration into an electronic signal. After almost 40years of NASA and DOD funding, the slow-scan, scientific-grade,charge-coupled device (CCD) camera is now an almost perfectsolution to this problem.

Success in modern biological light microscopy depends to anever-increasing extent on the performance of CCD cameras.Because such cameras differ widely in their capabilities and arealso items that most biologists buy separately, rather than as partof a system, some knowledge of their operation may be useful tothose practicing biologists who have not yet found it necessary tobe particularly interested in “electronics.” Although the basics ofCCD operation are described in many other chapters (particularly,Chapters 4, 10 and 12) this Appendix describes the operating principles of these devices in greater detail and also discusses theways that they “don’t work as planned.” It then covers the opera-tion of the electron-multiplier CCD (EM-CCD), a new variant that reduces the read noise almost to zero, although at the cost of reduced effective quantum efficiency (QEeff).1 The secondsection, How to choose a CCD, is a review of CCD specificationswith comments on the relevance of each in fluorescencemicroscopy.

PART I: HOW CHARGE-COUPLED DEVICES WORK

The first step is to imagine a rectangular area of the Si surface asbeing divided into rows and columns, or more usually, lines andpixels. Each pixel is between 4 ¥ 4mm and about 24 ¥ 24mm insize and the location of any pixel of the surface can be defined interms of it being x pixels from the left side, on line y.

To construct an actual system like this, start with a smooth Sisurface; cover it with a thin, transparent, insulating layer of SiO2;deposit onto the SiO2, a pattern of horizontal strips, made out of atransparent conductor called amorphous silicon (or poly-silicon),so that the strips cover the entire image sensor area. Although,viewed from the top, these strips partially overlap each other, they

are kept electrically separate from their neighbors by additionallayers of SiO2. Every third stripe is connected together to formthree sets of interdigitating strips that we will refer to as Phases 1,2 and 3 (f1, f2, f3, Fig. A3.1). Taken together, all these phases con-stitute the vertical register (VR) and, after the assembly has beenexposed to a pattern of light, they are used to transfer the photo-induced charge pattern downwards, one line at a time. The pixelsalong each line are separated from each other by vertical strips ofpositively doped material injected into the Si. These positive“channel blocks” create fields that prevent charge from diffusingsideways without reducing the active area of the sensor.

Any photon that passes through the stripes and the SiO2, isabsorbed in the Si, producing a PE. If a small positive voltage (~15volts) is applied to the f1 electrodes, any PE produced nearby willbe attracted to a location just below the nearest f1 strip (Fig. A3.2).As additional PEs are produced, they form a small cloud of PEsreferred to as a charge packet. The number of PEs in the packet isproportional to the local light intensity times the exposure periodand the problem now is to convey this packet to some locationwhere its size can be measured, and to do this without changing itor losing track of the location from which it was collected. Thiswill be achieved by using the overlying electrodes to drag thecharge packet around in an orderly way until it is deposited at thereadout node of the charge amplifier.

Charge CouplingThe dragging mechanism operates in the following way: First f2

is also made positive so that the cloud diffuses to fill the areaunderneath both f1 and f2. Then f1 is made zero, forcing the packetto concentrate under f2 alone (Fig A3.2).

So far, these 3 steps have succeeded in moving the chargepackets that were originally under each of the f1 electrodes down-wards by one phase or 1/3 of a “line” in the x-y raster. If thissequence is now repeated, but between f2 and f3 and then againbetween f3 and the f1 belonging to the next triplet of strips, packetswill have moved down by the one entire raster line. PEs createdwithin a particular pixel of each horizontal stripe remain confinedby the channel stops as they are transferred to the next line below.

A pixel of the image is therefore defined as the area under atriplet of overlying, vertical charge-transfer electrodes andbetween two neighboring channel blocks. The pixels on scientificCCDs, are usually square, 4 to 30 mm on a side while those oncommercial, video CCDs are likely to be wider than they are high,to conform with the reduced horizontal resolution of commercialvideo standards. Only square pixels can be conveniently displayedin a truly digital manner. Larger pixels have more leakage current(dark-current), but are also able to store more charge per pixel (seeBlooming, below).

Appendix 3

More Than You Ever Really Wanted to Know AboutCharge-Coupled Devices

James B. Pawley


1 This loss can be avoided if the system is used in photon-conting mode.

James B. Pawley • University of Wisconsin, Madison, Wisconsin 53706

More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 919

Drive pulseconnections

23

Verticalphase One pixel Control electrodes

Horizontal phaseDrive pulse connections

Readoutsection

Imagesection

Output

BASIC CCD ARRAY

1

Channel stop

Parallel channel

Serial channel

Readout node

Φ

Φ4

Φ

Φ

Φ5 Φ6

FIGURE A3.1. Layout of CCD array, viewed en face.

A

B

C

FIGURE A3.2. Charge coupling: Three stages in theprocess of moving a charge packet initially beneathphase 1 (A), so that it first spreads to be also under phase2 (B) and finally is confined to entirely under phase 2(C). These 3 steps must be repeated 3 times before thecharge packet has been moved downwards (or in thediagram, to the right) by one line of the CCD array.


At the bottom of the sensor, an entire line of charge packets issimultaneously transferred to the adjacent pixels of the horizontalregister (HR, also sometimes called a shift register). Like the VR,the HR is composed of a system of overlying poly-Si electrodesand channel stops. Each column of pixels in the VR is eventuallytransferred directly into the same specific pixel on the HR. Thethree phases of the HR (f4, f5, f6) work exactly like those in theVR, except that they must cycle at a much faster rate because theentire HR must be emptied before the next line of packets is trans-ferred down from the bottom line of the VR. In other words, in thetime between one complete line-transfer cycle of the VR and thenext, the horizontal register must cycle as many times as there arepixels in each line.

At the right-hand end of the HR is a charge amplifier that mea-sures the charge in each packet as it is transferred into it from thelast pixel of the HR. The first pixel to be read out is that on theextreme right-hand side of the bottom line. The last pixel will bethat on the left side of the top line.2

The entire charge-transfer process has the effect of codingposition as time. If we digitize the signals from the charge ampli-fier, and store the resulting numbers in a video memory, we willbe able to see a representation of the light intensity pattern strik-ing the sensor on any monitor attached to this video memory. Alter-natively, as long as the dimensions of the CCD array match thoseof some video standard, such as NTSC or PAL, the time sequenceof charge-packet readout voltages can be smoothed and, with theaddition of synch pulses, turned into an analog video signal. Whilethis latter process is often convenient, it is a poor plan if the analogsignal must then be re-digitized. The necessity to digitize twice canreduce the effective horizontal resolution of the CCD sensor byabout a factor of 2 and because the process is AC coupled, photo-metric accuracy is severely compromised.

It is important to understand the relationship between thecharge-transfer electrodes and the charge packet. The electrodesdo not somehow “connect to” the charge packet, and “conduct” itto the amplifier. Such a process would be subject to resistive losses,charge would be lost and a lot of “wires” would be needed. Thecharge-coupling process is better thought of in terms of a ballbearing “dragged” over the surface of a loose blanket by movinga cooking pot around underneath the blanket. The weight of theball and the lip of the pot create a dimple and gravity keeps theball in the dimple as the pot is moved. The voltage on the charge-transfer electrode creates an electronic “dimple.” Changing thevoltages on nearby electrodes moves the dimple. In this way,groups of charged particles (electrons) can be pushed aroundwithout actually “touching” or losing them.

Readout MethodsThere are three distinct methods for reading out the charge patternof a CCD: full-frame, full-frame transfer and interline transfer(Fig. A3.4). Most early scientific CCDs used the first method,which operates as has just been described. Although full-framereadout provides the largest sensitive area for a given area ofsilicon, the lowest level of readout noise and the greatest photo-metric accuracy, it also has some disadvantages. One cannot bothcollect and read out signal at the same time. Unless some sort ofshutter is used to prevent light from striking the sensor during ver-tical transfer, signal will be added to any packets that are trans-

ferred past bright features in the image, producing vertical streak-ing. This problem is more important when the exposure time isshort relative to the readout time.

In frame transfer readout, at the end of the exposure, theentire charge pattern is rapidly (0.1–3 ms) transferred by charge-coupling to a second 2D storage array. The storage array is thesame size as the sensor array and is located next to it but it is phys-ically masked with evaporated metal to shield it from light. Thecharge pattern is then read out from the storage array while thesensor array collects a new image. Because vertical transfer can bemuch faster if the charge packets do not have to be read out, thissystem reduces streaking by up to 1000¥ but does not eliminate itand the need for a storage register reduces the fraction of the Sisurface area that can be used for sensing by 50%.

In interline transfer, the masked storage cells are interlacedbetween the sensor cells (i.e., each pixel is divided into sense andread areas). After exposure, all charge packets can be moved to thereadout array in less than a microsecond. This ability can be usedas an electronic shutter to eliminate vertical smearing but, becauseat least half of the area of each sensor must be masked, and anylight striking a masked area is lost, the “fill factor” of the sensor isreduced, proportionately decreasing QEeff. A solution to the “fill-factor” dilemma is to incorporate an array of microlenses, alignedso that there is one above every pixel. With such a system, most ofthe light striking any pixel will be focused onto the unmasked area.3

Although microlenses restore the QEeff somewhat, the full-wellsignal possible is still limited by the smaller sensitive area.

WHAT COULD GO WRONG?

When I first heard the CCD story, it struck me as pretty prepos-terous! How could you get all the correct voltages (9 differentvoltage combinations per pixel shift, ~3.6 million for each TVframe, 108 million/s for video rate!) to the right charge-transferelectrodes at the correct times? How could you get all of the chargein a packet to stay together during a transfer? Wouldn’t Poissonstatistics apply, making even one transfer imprecise and the 2000transfers needed to read out the top, right pixel of a 1000 ¥ 1000pixel array impossibly inaccurate? How long would the PEs stayfree to be dragged around the lattice? Wouldn’t the charge packetsdecay with time?

In fact, many of these problems did occur, but remedies to mosthave now been devised. The difference between a $300 commer-cial CCD camera and a $65,000, top-of-the-line scientific CCD canoften be measured in terms of how many of these remedies havebeen implemented. Therefore, it is worthwhile trying to understandsome of them so that one can buy what one needs. The followingdiscussion will define and discuss some of the more importantCCD technical specifications.

Quantum EfficiencyQuantum efficiency is the ratio of the number of impingingphotons to the number of PEs produced.4 Any photon with energyin the range of 1–100 eV striking crystalline Si has a very highprobability of producing a PE. However, reflections and absorp-tion by the overlying polysilicon electrodes,5 reduce the QE of

2 This may seem backwards until one remembers that any image of the realworld is usually focused onto the CCD by a single, converging lens, a processthat always inverts the image.

3 This occurs only as long as the initial angle of incidence is near to normal, acondition met when CCDs are used for light microscopy.

4 In the visible range, each absorbed photon makes only one PE.5 Kodak had pioneered the use of charge transfer electrodes made out of In and

Sn oxides that scatter less light than do those made of poly-Si.


front-illuminated CCDs especially in the blue end of the spectrum.To reduce this effect, some UV-enhanced sensors are coated withfluorescent plastics, which absorb in the blue and emit at longerwavelengths. Others have their backs etched away and are turnedover to permit the illumination to reach the light-sensitive areafrom the back side.6 Figure A3.4 shows the intrinsic QE of differ-ent types of CCD (not Qeff, which would take into account the lightlost if some of the sensor is covered by charge storage areas). Theeffective QE can usually only be determined by actual measure-ment or by very careful evaluation of the published specifications(QEeffective = QEintrinsic ¥ fill factor).

Edge EffectsIn early CCDs, PEs were often “lost” in the crystalline imperfec-tions that are always present at the Si/SiO2 junction. To avoid this,ion implantation is now used to make an N-doped, sub-surfacelayer called the buried channel about 1 mm below this surface (Fig.A3.2). This channel attracts the free PEs, keeping them away fromthe edge of the Si crystal. Any serious CCDs will have a buriedchannel but the need for ion-implantation keeps CCD chip priceshigh! Figure A3.5 shows the readout noise, in root-mean squared(RMS) electrons/pixel, for surface and buried-channel CCDshaving two different pixel sizes. From this you can see that smallpixels (here ~5.5 ¥ 5.5mm) have lower read noise than larger ones(~17 ¥ 17mm), mostly because the larger ones have higher capac-itance and capacitance is the most important parameter of read-

amplifier noise. One can also see that at readout speeds higher than1MHz (or 1 second to read out a 1024 ¥ 1024 CCD), the readnoise increases with the square root of the read speed.

Charge LossThe lifetime of a PE (before it drops back into the ground state)depends on the purity and crystalline perfection of the Si and onother factors such as temperature. Generally it is long enough thatlittle charge is lost during the exposure times commonly used influorescence microscopy. If necessary, it can be increased bycooling the detector, something often done to reduce dark charge.

Leakage or “Dark Charge”Dark charge is the charge that leaks into a pixel during the expo-sure time in the absence of light. It can be thought of as the darkcurrent7 deposited into one pixel. Many processes other thanphoton absorption can add PE to the charge packet. The magni-tude of this dark charge depends on the length of the exposure, andis substantially reduced by cooling. The rule of thumb is that forevery 8°C of cooling, the dark charge is halved. As noted above,dark charge is principally a problem because it produces Poissonnoise equal to the square root of its magnitude, and if this is left unchecked, it can significantly increase the noise floor of theCCD.

Since ~1987, a process called multipinned phasing (MPP) hasbeen available to reduce dark charge build-up by about a factor of1000, making it immeasurable in exposures up to a minute or so.This feature should be specified if one expects to use exposureslonger than a few seconds without deep-cooling.

FIGURE A3.3. Four CCD readout patterns: Full-frame,frame-transfer, interline transfer and gain register (EM-CCD).

6 Back-illuminated CCDs have to be thinned to 7–10 mm so that conductionelectrons created near what would have been the back surface can respond tothe fields created by the buried channel and the CC electrodes. Thinningincreases cost and also reduces QE at longer wavelength where the absorp-tion distance of the photons becomes comparable with the actual thickness.Back-illuminated CCDs are also more expensive because it is difficult tocreate electrical contacts with electrodes, etc., that are now on the bottom sideof the chip.

7 A current is a flow of charge measured in charge/time. The unit of charge isthe Coulomb (c). The unit of current is the Ampere (A). One Amp representsa flow of one Coulomb/s or 6.16 ¥ 1018 electrons/s.


It should also be remembered that, while dark charge is nevergood, its average value can be measured and subtracted on a pixel-by-pixel basis, by subtracting a “dark image” from each recordedimage as part of flat-fielding. However, because, by definition“dark” images contain very few photons/pixel, they have relativelyhigh Poisson noise and low S/N. Therefore, a number of suchimages must be averaged to produce a correction mask that is sta-tistically defined well enough that subtracting it from the data doesnot substantially increase the noise present in the final, correctedimage.

This is not a problem when there are many counts in each pixelbecause the subtractive process of dark-charge normalizationinvolves a change that is small compared with the intrinsic noisepresent in a large signal. It can be a problem when the black maskimage is subtracted from a faint image that also contains only afew counts/pixel.

What cannot be removed by flat-fielding is the Poisson noiseassociated with the dark charge. This is equal to the square root of

the number of electrons/pixel it represents. CCDs should alwaysbe operated such that the noise on the dark charge is less than thereadout noise. On conventional CCDs this condition can usuallybe met quite easily by slightly cooling the sensor (0°C or about -20°C from ambient). The use of lower temperatures is compli-cated by the risk of condensing atmospheric water, a process thatcan be avoided only by enclosing the sensor in a vacuum chamber.Generally, a vacuum-hermetic enclosure, combined with good outgassing prevention, carries with it the significant benefits ofmore effective cooling, long-term protection of the sensor frommoisture and other degrading organic condensates as well as the prevention of front-window fogging. At video rate, whereexposures are short, dark charge is only a problem when thereadout noise is reduced to <1e/pixel, as it is when an “electron-multiplier” (EM) charge amplifier is used (see below and alsoChapters 4 and 10). In EM-CCDs the read noise is so low that darkcurrent becomes the main source of noise and cooling to -80°Cbecomes necessary.

FIGURE A3.4. Intrinsic QE as a function of wave-length for a front-illuminated CCD (blue), a visible-enhanced, back-illuminated CCD (green) and aUV-enhanced CCD (red).

1

2

3

5

10

20

30

50

100

10 ns 100 ns 1 µs 10 µs 100 µs

A = 30 µm2

A = 300 µm2

A = 30 µm2

A = 300 µm2

Measured

Surface channel

Surface channel

Buried channel (T<200K)

Buried channel (T<200K)

Clamp-to-sample time

Readout noise, electrons, rms

10 MHz 1 MHz 100 kHz 10 kHz100 MHz Pixel readout rate

FIGURE A3.5. CCD field effect transistor (FET)noise as a function of pixel dwell time for large andsmall pixels and when using buried channel vs. surfacechannels. Smaller pixels have less read noise becausethey have less capacitance. Buried channels have almost10¥ less read noise than surface channels.


BloomingAs more photons are absorbed, the charge packet clustered aroundthe buried channel grows and mutual repulsion between these electrons renders the field imposed by the charge-transfer electrodeever less successful in keeping the packet together. The maximumcharge packet that can be stored without it overflowing into nearbypixels can be estimated by multiplying the pixel area (in squaremicrometers) by 600PE/pixel (i.e., 27kPEs for a 6.7 ¥ 6.7mmpixel, 540kPEs for a 30 ¥ 30mm pixel). This overflow problem isreferred to as “blooming” and, in CCDs for the home-video marketit is limited by the presence of an n-layer, deeper in the Si. Whenthe charge packet gets too big, mutual repulsion between the PEsforces some of them into this overflow layer, through which theyare conducted to ground.

While this anti-blooming feature is convenient for removingthe effects of the specular reflections found in images from everyday life,8 it is not incorporated into many full-frame or frame-transfer scientific CCDs because it reduces QE for long-wavelength light. As this light penetrates farther into the Si crystalbefore being absorbed, much of it reaches the overflow layer whereany PEs produced are lost.

Incomplete Charge TransferSometimes, an imperfection in the Si will produce a pixel that“leaks” charge. Charge deposited into, or transferred through, thispixel will be lost, producing a dark vertical line above it. In addi-tion, if one pushes the pixel clock too fast, some PE in the packetwill not move fast enough and they will be left behind. In generalhowever, on a slow-scanned, scientific CCD, fewer than 5PEs outof a million are lost (or gained) in each, slower, vertical transferand only 50 (0.005%) are lost during each, faster horizontal trans-fer. In such devices, the main noise term is Poisson noise for anysignal level above ~20PE/pixel,9 and it seems hard to imaginedoing much better than this except for signal levels <16 PE/pixel.

On the other hand, it is also true that the vast majority of CCDsmade (those for camcorders, surveillance cameras and even many

scientific applications), operate with much (100¥?) less perfection.In microscopy today, we find CCDs that span this range of per-formance (Table A3.1).

All CCDs are not equal!!

CHARGE AMPLIFIERS

So far, I have described an image sensor in which up to 90% ofthe impinging photons make free PEs and explained how thecharge packets that result from many photons hitting a given pixelcan be conveyed to the charge amplifier, in a time-labeled mannerand almost without change. Clearly the performance of the entireimage detector will depend crucially on the capabilities of thisamplifier.

What Is a Charge Amplifier?Although most scientists have had some exposure to electronic cir-cuits that amplify an input voltage or current, they may be lessfamiliar with the operation of the type of charge amplifier foundin a CCD. The following outline is presented to enable the readerto understand enough about the process to appreciate some of theimportant differences between the various types of CCD.

Because of the pulsatile nature of the CCD charge deliverysystem, the optimal way to measure charge packet size is to depositit into a (very) small capacitor (the “read node”) and then measurethe voltage on this capacitor with a high impedance amplifier. Asa field-effect transistor (FET) has an almost-infinite input imped-ance, it is ideal for this purpose and in fact, its existence makescharge-amplification possible.

There are two basic types of conventional CCD readout amplifier, non-destructive and destructive.10 Both employ FETamplifiers.

Non-destructive (“skipper”) amplifiers use an FET with a“floating gate” to sense the size of a charge packet by respondingto the moving field that is produced as the packet is transferredalong a charge-coupled register. Because the charge packet itselfis not affected by this process, the process can be repeated hun-dreds or even thousands of times. If the results of all these mea-surements are averaged, very low readout noise levels (>±1electron/pixel) can be obtained, but at the cost of a substantial

8 Features such as the image of the sun reflecting off a shiny automobile canbe over 1,000¥ brighter than the rest of the scene. Fortunately, such extremelybright features are seldom found in microscopic images unless a crystal offluorescent dye occurs in the field of view.

9 This calculation assumes that the read noise is 4e/pixel, and this will be lessthan the Poisson Noise for any signal >16 PE. However, as many CCDs usedin microscopy have >4e/pixel of noise, this cut-off point should not be con-sidered inflexible.

TABLE A3.1. Typical Performance of Various Types of CCD Cameras. The “Sensitivity” Column Is a Reasonable Estimate ofthe Relative Suitability of the Camera for Detecting Very Faint Signals. It Spans a Very Large Range of Performance!

All CCDs are not equal

Type Grade QE % (effective) Noise (e/pix) Sensitivity (relative) Bit depth Dynamic Range

Video commercial color 10 200 1 10 1,000monochrome 20 200 2 10 1,000

Digital 1Mhz, color 15 50 12 12 4,0001Mhz, mono 30 50 24 12 4,000Back. Illum/ 90 5 720 15 40,000

slow-scanLLL-CCD 45 0.1 (18,000)* ?* 200,000

(EMCCD)

*Because the gain of the electron multiplier amplifier is unknown and large, it is not simple to measure, or even define, the sensitivity and bit depth of the EM-CCDs.

10 The “electron-multiplier” amplifiers mentioned previously, act essentially aspre-amps to the conventional FET amps described here. They will be coveredlater in this Appendix.


(¥100 or ¥1,000) increase in readout time and logical complexity.This approach might make sense on a Mars probe but it has notbeen used in microscopy to my knowledge.

Destructive readout amplifiers are more common, probablybecause they can operate more rapidly (Fig. A3.6). As imple-mented in a scientific CCD, the charge amplifier consists of thefollowing components:

• overlying charge transfer electrodes to drag the next chargepacket into the “read node”

• the read node itself: a 0.03–0.1 picofarad capacitor• the sense FET• the reset FET

In operation, fields from the overlying charge-coupling elec-trodes force a charge packet into the read-node capacitor, creatinga voltage, Vc, that is proportional to the amount of charge in thepacket. This voltage is sensed by the sense FET and the output ispassed, via additional amplifiers, to the analog-to-digital converter(ADC) where the signal is converted into a digital number. Finally,just before the next charge packet is coupled into the read node, areset FET discharges the capacitor, forcing Vc to zero, and allow-ing the read-FET to sense it again.

FET Amplifier PerformanceThe signal current (signal charge/s) coming from a CCD sensor isvery small. Suppose that there were, on average, 400 PE in everypixel of a 512 ¥ 512 pixel sensor.11 Reading this out in one secondwould constitute a current of only 10-11 Amps. The current throughthe bulb in a home flashlight is 1010 times more. A very good con-ventional electronic amplifier designed to amplify this current

would, itself, produce a random electronic noise signal larger thanthis, and electronic noise increases with readout speed, read-nodecapacitance and, to a lesser extent, temperature.

The success of the CCD in overcoming this limitation dependson two factors:

• The extremely small capacitance of the read node comparedto that of any other photosensor such as a photodiode.

• Special measurement techniques such as correlated double-sampling

Clearly there are a lot of tricks to making the perfect CCDamplifier and not all CCDs employ them. Table A3.1 lists typicalperformance for a variety of common camera types.

NOISE SOURCES IN THE CHARGE-COUPLED DEVICE

Fixed Pattern NoiseWhen exposed to a uniform level of illumination, some pixels ina CCD array will collect more charge than others because of smalldifferences in their geometry or their electrical properties. Conse-quently, it can be necessary to use stored measurements of the rel-ative sensitivity of each pixel to normalize, or “flat field,” the finaldataset on a pixel-by-pixel basis. This is accomplished by firstrecording an image of a featureless “white” field. This is oftenapproximated by a brightfield transmission image with no speci-men, a process that will also record “inhomogeneity,” or mottle,in the optical system. Differences in gain between pixels areevident as visible as nonuniformities in the digital signal stored inthe memory and these are used to derive multiplicative correctioncoefficients.12

Unfortunately, one can only preserve the high precision of theCCD output if the coefficient used to normalize each pixel isequally precise. In any event, these correction coefficients varywith both the photon wavelength and the angle at which the lightpasses through the polysilicon electrodes on its way to the buriedchannel. This, in turn, depends on the details of the precise opticalpath in operation when an image is recorded and may even changewith microscope focus! As the intrinsic noise of a pixel holding360k, PEs is only ±600 electrons or 0.16%, pixel-to-pixel nor-malization for changes in sensor gain is seldom perfectly effectiveand consequently there is usually some level of “Fixed-patternnoise” superimposed on the final data.

In addition, the “white” image that must be used for pixel-levelsensitivity normalization is itself subject to intrinsic noise (±600electrons for a signal from a pixel with a full well charge of 360kelectrons) and so multiplicative normalization may actually addsome noise to the raw, uncorrected signal! Fortunately, if the whiteimage can be defined by a multi-frame averages of several, nearlyfull-well “white” images, this normalization noise should only benoticeable when the image data to be corrected is similarly noisefree. Without details of the signal levels present or the opticalsystem in use, it is difficult to estimate the magnitude of normal-ization noise but it will be comparatively less important for imagesof faint objects containing few counts/pixel because these mea-surements are themselves less precise.

Supply voltageVReset

Resettransistor

Chargeinput

∆V = Q

Readout transistor, Gain G

Output, G∆V = GQ

LoadQ

CCD Register Substrate and ground

Resettrigger

Cn

Readout node capacitance

Cn

FIGURE A3.6. Destructive read-out amplifier for a CCD chip.

11 Although this number may seem small, it is actually quite high compared tosome uses in biological confocal microscopy. Many authors have found thatin normal” use, a single-beam confocal microscope used to image a fairlyfaint stain will count 4–8 PE/pixel in bright areas of the image. Allowing thatthe effective QR of a good CCD will be ~10¥ higher than that of the photo-multiplier tube used in the confocal microscope, this makes the expectedpeak CCD signal in an image from a disk-scanning confocal microscope only40–80 PE/pixel.

12 These correction coefficients are small and only needed when operating onimages involving large numbers of photons (and consequently having rela-tively low Poisson noise and good S/N).


It should be also noted that the vignetting and “mottle” visiblein images characteristic of video-enhanced contrast microscopywill produce small intensity errors in the data obtained by bothwidefield and confocal. However, this noise term will be morenoticeable in widefield where more photons are used and hence theprecision of the data is greater. Mottle is produced by dirt andsurface imperfections on any optical components that are notlocated exactly at aperture planes, as well as by non-uniformitiesin the image sensor. Fortunately, to the extent that it is stable withtime, mottle will be removed by the flat-field correction for CCDsensitivity just discussed.

What will not be removed is any change in signal caused bystray light (room light, light that goes through filters designed toremove it, etc.). The simplest test of any CCD set-up is to recordan image of “nothing” (i.e., room dark, no excitation, no specimenetc.). Then do the same with 100¥ longer exposure time with theroom lights at your normal operating level. Now adjust the displaylook-up tables so that you can “see the noise” in both the imageson the screen. Although the only difference between the twoimages should be increased dark noise in the image with the longerexposure, this is seldom the case.

Noise from the Charge AmplifierNoise is generated by both the readout and the reset FETs in thecharge amplifier. Noise generated in the readout FET reaches theADC directly. If thermal noise in the reset FET prevents it fromcompletely discharging the read-node capacitance, it produces arandom offset at each pixel (i.e., the read-node voltage is not resetexactly to zero). This is referred to as Reset Noise and has theeffect that the dark charge seems to vary from pixel to pixel. For-tunately, Reset noise can be almost eliminated by employing thetechnique of Correlated Double-sampling (CDS) in the readoutamplifier. In CDS, the circuitry of the charge-to-voltage amplifieris modified so that the output is proportional to the differencebetween the value of Vc just after the reset pulse and its value afterthe next charge packet has been inserted.

Although CDS essentially eliminates the effect of reset noise,it also distorts the noise spectrum. On the one hand, this distortionhas the beneficial effect of converting the low frequency, 1/F noisefrom the FET into broadband noise which is more easily treatedtheoretically and which is less visually distracting than the short,horizontal flashes characteristic of 1/F noise.13 On the other hand,it means that the input to the ADC must be carefully frequency-filtered. This filtering can be implemented either by employing RCcircuits or by using dual-slope integration (DSI) in the ADC itself.If there are large intensity variations between neighboring pixels,the use of RC circuits will effectively compromise the largedynamic range of the CCD. Therefore, ADCs using DSI areemployed on most slow-scan scientific, cooled-CCDs.

The fact that CDS and, in particular, DSI work best at lowreadout speeds is a final reason why most scientific CCDs operatebest at relatively low readout speeds (Fig. A3.5). The other tworeasons are improved charge transfer efficiency and the reductionin broadband electronic noise from the FETs (noted above.)

Where Is Zero?A final important feature of the CCD readout is that, compared tothe photomultiplier tube (PMT), it is relatively difficult to deter-mine the exact output signal level that corresponds to a zero-lightsignal. A properly operated PMT never records negative counts.However, as the electronic readout noise of a cooled-CCD is anRMS function with both positive and negative excursions, therewill be some pixels that measure lower than the mean value of thezero-light pixel intensity distribution.

To ensure that no data is “lost,” scientific CCDs are usually setup so that the zero-light signal is stored to be a few tens of digitalunits (ADU) above zero. A histogram of numbers stored from a“black” image will show a Gaussian-like peak centered at theoffset and with a half-width equal to 2¥ the RMS read noise (seeFig. 4.20). This offset makes it more difficult to apply the gain andoffset normalization procedures to images that record only a fewdetected photons in each pixel, a factor that will become moreimportant as CCDs are increasingly used to image living cells thatcannot tolerate intense illumination and which therefore producesubstantially lower signal levels.

A NEW IDEA: THE GAIN REGISTER AMPLIFIER!!

Early in 2002, a new type of readout amplifier was introduced byTexas Instruments (Houston, TX) and E2V Technologies (Chelms-ford, UK). As only E2V makes back-illuminated sensors, I willdescribe their system but both work along similar lines. E2V orig-inally referred to their device as the “gain register” and its purposeis to amplify the size of the charge packet before it arrives at theread node. Although the term gain register has recently beenreplaced by the term “electron multiplier”, it is important toremember that these new detectors work on a completely differentprinciple from that employed in intensified-CCDs.

The gain register superficially resembles an additional HR,with two important differences:

• There are 4 phases rather than the usual 3 and the new phase consists of a grounded electrode located between f1

and f2.• The charge transfer voltage on f2, is now variable, between

+35 and +40 volts rather than the usual +15 volts.

As a result, when f2 is excited, there exists a high electric fieldbetween it and the grounded electrode. The high field acceleratesthe electrons in the charge packets more rapidly as they pass fromf1 to f2 with the result that each PE has a small (but finite; usuallyin the range of 0.5% to 1.5%) chance of colliding with a latticeelectron and knocking it into the conduction band (Fig. A3.7).Assuming the 1% gain figure, this means that for every 100 PE inthe packet, on average one of these will become two electronsbefore it reaches the space under f2. Although this seems like atrivial improvement, after it has been repeated as part of the 400to 590 transfers in the gain register, a total average gain of hun-dreds or even thousands is possible. If the voltage on f2 is reducedto normal levels, the sensor operates as a normal CCD.

As a result, a single PE can be amplified sufficiently to besafely above the noise of the FET amplifier, even when it is oper-ating at speeds considerably higher than video rate (35–50MHz,vs. 13MHz for video). As the amount of gain depends exponen-tially on the exact voltage on f2, it is possible to “dial in” theamount of gain needed to keep the signal level well above the noiseof the FET amplifier. However, it is important to remember that

13 In a CCD without CDS, noise features will seem to be smeared sideways,while in one with CDS, they will appear as one-pixel-wide stipple with nodirectionality.


the use of high EM gain will tend to saturate the “full-well” capac-ity of later pixels in the gain register, reducing intra-scene dynamicrange.14 Although this effect can be reduced to some extent bymaking each pixel in the gain register (and the read node) larger,this approach is limited by the fact that one triplet of electrodescan control a band of silicon only ~18mm wide and because alarger read-node capacitance increases the read noise of the FETamplifier.

In sum, the gain-register CCD works like a normal fast-scanCCD with no read noise. The high scan speed makes focusing andsample scanning quick and easy and the device preserves the fullspatial resolution of the CCD because the charge packet from onepixel is always handled as a discrete entity (unlike in an intensi-fied-CCD). Of course, with fast readout, there is less time to accu-mulate much signal and the resulting image may have considerablePoisson noise. But this is not the camera’s fault!

Alternatively, the output of many frames can be summed toreduce Poisson noise or, if the signal is bright, one can turn off theEM gain and have a fully functional scientific CCD.15

If the gain-register CCD is read out fast, there is so little timefor dark charge to accumulate that cooling would seem unneces-sary until one remembers that one can now “see” even one PE ofdark-charge above the read noise. Because multi-pinned phasing(MPP) is less effective during the readout clockings, significantdark charge can be generated during readout. If the exposures areshort, this source of dark charge becomes significant, and in anEM-CCD, even one electron is significant! In practice, the bestperformance is obtained when the EM-CCD is cooled to between-80° and -100°C.

EM-CCDs have one other important form of “dark noise”called Clock Induced Charge (CIC, also known as spurious noise).CIC typically consists of the single-electron events that are presentin any CCD, and are generated by the vertical clocking of chargeduring the sensor readout. The process involved is actually thesame impact ionization that produces multiplication in the gainregister; however, levels are much lower because lower voltagesare involved. In conventional CCDs, CIC is rarely an issue assingle-electron events are lost in the read noise. However, in EM-CCDs where the read noise is essentially zero and dark charge hasbeen eliminated through effective cooling, CIC is the remainingsource of single-electron, EM-amplified noise. If left unchecked,it can be as high as 1 event in every 7 pixels. Fortunately, it canbe minimized by careful control of clocking voltages and by opti-mizing the readout process to cope with faster vertical clock speeds(down to 0.4ms/shift). This leaves a detector with less than onenoise pulse in every 250 pixels: a detector extremely well adaptedfor measuring zero!

Of Course, There Is One Snag!The charge amplification process is not quite noise free becausethe exact amount by which each electron in the packet is ampli-fied varies in a stochastic manner (i.e., some electrons are “moreequal” than others.). The statistical arguments are discussed in apaper found at the URL listed below and in Chapter 4. In summary:as the multiplicative noise inherent in the charge multiplicationprocess creates noise that has a form very similar to that producedby Poisson statistics, the easiest way to think of its effect is toassume that the amplifier has no noise at all but that the signalbeing fed into it is half as big as it really is. In other words, thecamera will work perfectly but it will work as though it has a QEthat is only half of what it really is. Back-illuminated sensors arenow available with an intrinsic QE of about ~90% or ~45% whenused in the gain-register mode. This is 5–10¥ better than the per-formance available from most PMTs especially in the red end ofthe spectrum.

It is worth noting that one can use electron multiplication andstill maintain the full QE by using the detector in photon-countingmode, as is now being done by many astronomers. Photon count-ing is only possible when one is able to confidently see a single-

FIGURE A3.7. Energy diagram of an electron-multiplier CCD amplifier. The high field region that occurs between f2 and fDC when f2 goes strongly posi-tive (right) causes about 1% of the electrons passing this region to collide with a lattice electron with sufficient energy to boost it to the conduction band. Repeatedover hundreds of transfers, this process is capable of providing an average amplification of hundreds or even thousands of times.

14 If a register designed with enough pixel area to hold a normal full-well chargeof 30,000 electrons, is used with a gain of 100¥, then the pixels near the endof the gain register will become full whenever the original charge packet has>300 electrons.

15 Because, as noted above, the read node of the FET amplifier at the end ofthe gain register in an EM-CCD has a relatively large capacitance, E2V offerstwo separate FET readout amps. The one mounted at the end of the gain reg-ister is optimized for fast readout. The other is mounted at the end of the HRnot connected to the gain-register, has low input capacitance and is optimizedto read out slowly with low noise. Signal is sent to the latter by reversing thecharge transfer sequence applied to the HR.


photon event as different from any dark event and when thenumber of photons collected during an exposure is so low thatthere is little probability of >2 photons falling into a pixel. Toimplement photon-counting, one records a sequence of short expo-sures containing “binary”-type single-photon data, and combinesthem to generate an image that is free of multiplication noise.16 Tobe useful for recording dynamic events in living cells, an extremelyfast frame rate would be needed. This may be more possible withsome future EMCCD sensors.

(More info on EM-CCDs at http://www.emccd.com andhttp://www.marconitech.com/ccds/lllccd/technology.html)

PART II: EVALUATING A CHARGE-COUPLED DEVICE

A. Important Charge-Coupled Device Specs forLive-Cell Stuff!Although in Part I, much time was spent discussing cooled, slow-scan, scientific CCDs, in fact, these have not been much used inbiological microscopy since Sony introduced the ICX085, 1k ¥1.3k, micro-lens-coupled, interline-transfer chip in the late 1990s.Although initially developed not for the scientific market but tomeet the needs of the Japanese high-definition TV standard, thesechips offered a set of practical advantages that biologists foundvery appealing:

— As an interline transfer chip, it needed no mechanical shutterand could be run so as to produce a continuous stream ofimages.

— The high readout speed (up to 20Mhz) allowed real-time im-aging compared with the 5–10s/frame readout then common.

— The 6.45 ¥ 6.45 mm pixels were small enough to sample theimage produced with high-NA 40¥ and 60¥ objectives.

— The 1k ¥ 1.3k raster size was both sufficient for most bio-logical microscopy and significantly higher than that of theother scientific chips then available.

— Mass production allowed the development of a micro-lensarray that increased the QEeff to an acceptable level for a front-illuminated, interline chip and did so at a price biologistscould afford.

As a result, the majority of CCDs sold for use in biologicalmicroscopy today use this chip or its higher-QE cousin, theICX285. Although mass production made quality CCDs availableto many who formerly could not have obtained one, it is impor-tant to remember that the read noise of ±8–24 electrons/pixel(depending on read speed) is substantially higher than ±2–3 elec-trons/pixel that characterized the best, slow-scan, scientific CCDs.Although, as noted below, the difference is only important if thedimmest pixel records fewer than ~50 electrons, and this seldomoccurs in widefield fluorescence microscopy, the disk-scanningconfocal fluorescence microscopes now available do provide animage in which this difference is significant (Chapter 10).

1. Quantum Efficiency (QE):QE is the ratio of photons striking the chip to electrons kicked intothe conduction band in the sensor. It should be at least 40% and

on back-illuminated chips, it can reach 90% (with somewhathigher fixed-pattern noise).

The fill-factor is the fraction of the sensor surface actually sen-sitive to light. On the best frame-transfer CCDs, it can be almost100%. On interline transfer CCDs it may be only 40%. Light notabsorbed in a sensitive area is lost, reducing the QEeff of the sensorproportionally.

Factors affecting QE:

Front-illuminated chips

• Light is scattered by the transparent, polysilicon charge-transfer electrodes that overlie the photosensitive siliconsurface.

• This scattering is more severe at shorter wavelengths. Lightthat is scattered is not detected.

• As blue light is absorbed nearer to the surface than red light,and “deep electrons” may go to the wrong pixel, CCD resolu-tion may be a bit lower than the pixel count at longer wave-lengths, especially on chips with small pixels.

• Best QE: ~20% blue, ~35% red/green• Two efforts to improve the QE of front-illuminated chips

include “Virtual Phase” (one phase “open,” Texas Instruments,Houston, TX) and the use of indium oxides for the transferelectrodes (Kodak, Rochester, NY). These have increased peakQE to the range of 55%.

Micro-lens array chips

• Sony has pioneered a process in which a micro-lens is mountedabove each pixel of a front-illuminated, interline-transferCCD. The lenses focus most of the impinging light onto a partof the CCD where reflection losses are least, pushing the QEto 65% in the green, less in the red (because of losses to theoverflow drain) and purple.

Back-illuminated chips

• Made by thinning the silicon and then turning it over so thatthe light approaches the pixel from what would have been theback side. This avoids scattering in the transfer electrodes andincreases the QE to about 90% in the green and >70% over thevisible range.

• More expensive because of the extra fabrication.• Slightly less resolution and more fixed-pattern noise, caused

by imperfections in the thinning operation, and the presenceof two sets of surface states.

Color Chips

• One-chip color sensors employ a pattern of colored filters, oneover each pixel. Light stopped by any such filter cannot bedetected and is therefore lost. The QE of such sensors is there-fore at least 3¥ lower than for an otherwise comparable mono-chrome chip.

• 3-chip color sensors use dichroic mirrors to separate the“white” light into three color bands, each of which is directedto a separate monochrome CCD sensor. While this would seemto ensure that “all photons were counted somewhere,” becausesuch systems seldom employ microlenses, their effective QEis not much better than the 1-chip color sensors and alignmentof the signal light is important.17

16 There is no multiplicative noise because any spike above the FET noise floorcounts as one electron, no matter how much it has been amplified.

17 While the QE is not much better, the resolution of the 3-chip camera is thesame as that of each chip, without the interpolation needed to disentangle the3 colored images from the output of a 1-chip color sensor.


• Color can be detected by making sequential exposures of amonochrome chip through colored glass or LCD filters. Thisproduces the same QE losses as the patterned filter but has the advantage that it can be removed when higher sensitivityis needed. This design is not suited for imaging movingobjects.

2. Readout Noise:This spec is a measure of the size of the pixels and the quality ofthe circuitry used for measuring the charge packet in each pixel.It is measured in “±RMS electrons of noise” (i.e., 67% of a seriesof “dark” readings will be ± this much).

• A good scientific CCD camera should have a noise level of <±5 electrons at a readout speed of 1M pixels/second.

• The readout noise increases with the square root of the readoutspeed (see Table A3.2).

• NO Free Lunch! A chip that has ±5e RMS of noise whenreadout at 100k pixels/sec (or 10 seconds to read out a 1024¥ 1024 chip), should produce ±50e RMS of noise if read outat 10M pixels/sec (or 0.1 sec to read the same chip).

What Is “Good Enough”?Very low readout noise is only essential when viewing very dimspecimens: luminescence, or low level fluorescence. Read noise isonly a limitation when it is more than the statistical noise on thephoton signal in the dimmest pixel (i.e., >sqrt of the number ofdetected photons = sqrt # electrons).

Consider the signal levels that you plan to use. Will the darkestimportant part of your image have zero signal or do you expectsome background signal from diffuse staining or out of focus light?If the dimmest pixel in your image represents ~100 electrons, thenthe Poisson or statistical noise on this background signal will be±10 electrons. “Adding” an additional ±10 electrons of readoutnoise will not make much difference to a measurement of thisbackground signal and it will be even less significant when addedto the even greater Poisson noise present in pixels where thestained parts of the image are recorded.

This is especially true because RMS noise signals add as the“sqrt of the sum of the squares” (i.e., the total noise from ±10 elec-trons of readout noise and ±10 electrons of Poisson noise is onlysqrt (100 + 100) = ±14 electrons).

On the other hand, if you are really trying to keep those cellsalive and you find that 2,000 electrons in the bright areas is enough,the dark areas may now be only 50 electrons. As the sqrt of 50 isabout ±7, an additional ±10 electrons of readout noise may nolonger be acceptable, but only if you have to make measurementsin the dark areas on your image. In this case, the obvious choiceis a slower, quieter CCD or an EM-CCD.

While in widefield fluoresecence, the background stain levelis seldom so low that the sqrt of the signal recorded is lower thanthe read noise, the disk-scanner does provide such an image(Chapter 10). As one of the main advantages of disk-scanning isthat one can scan an entire image plane very rapidly, the fact thatone can read out the EM-CCD very rapidly without increasing theread noise makes it the ideal detector for this type of scanner (or,indeed for high-speed line scanning confocal microscopes).

3. Pixel Size:

• Nyquist sampling: The size of a pixel on the CCD is, in itself,not very important BUT one must satisfy the Nyquist crite-rion: The pixels on the chip must be ~4+ smaller than thesmallest features focused onto it18 (see Chapter 4): Pixel size

on the chip determines the total specimen-to-chip magnifi-cation needed!

Two examples:

a. 1.4 NA 100¥ objective and a 1¥ phototube.• The Abbe Criterion resolution @ 400nm is about

0.22mm. Magnified by a total magnification of 100¥,this becomes 22 mm at the CCD.

• A CCD having 8 ¥ 8mm pixels samples such an imageadequately (~2.8 pixels/resolution element).

b. 1.3 NA 40¥ objective and a 1¥ phototube.• The Abbe Criterion resolution @ 400nm is now

0.25mm. Magnified 40¥ this becomes, 10 mm.• A CCD having 8 ¥ 8mm pixels is inadequate to sample

this lower-mag, high-resolution image.

If you must use this objective, you need either a higher magphototube (2.5¥) or a chip with 3 ¥ 3mm pixels or (as CCDpixels are seldom this small), some combination.

• Saturation signal level: The maximum amount of signal thatcan be stored in a pixel is fixed by its area. The proportion is600 electrons/square mm, so a 10mm ¥ 10mm pixel can storea maximum of 60,000 electrons before they start to bleed intoneighboring pixels. In practice, as fluorescent micrographs ofliving cells seldom produce signals this large, large pixels areusually unnecessary.

However, the saturation level also represents the top end ofanother spec, the dynamic range. This is usually quoted as 12-bit (4000 :1) or 14-bit (16,000 :1) etc., and represents theratio between the full-well saturation level and the readoutnoise. Therefore, a camera with relatively high readout noisecan still look good in terms of dynamic range if it has largepixels and hence a high full-well capacity. Conversely, a 12-bitcamera with small pixels can have less actual noise-per-pixelintensity measurement than a 14-bit camera with large pixels.

In this case, the noise level of the 14-bit camera is >3¥ thatof the 12-bit camera. Your signal/pixel would have to be 3¥ larger in order to be “seen” when using this particular 14-camera.

4. Array Size:19 The argument for small

• Assuming 0.1 mm pixels (referred to the object plane), a 512 ¥ 512 pixel chip will image an area of the specimen that isabout 51 ¥ 51 microns. If this is enough to cover the objectsyou need to see, this small chip has a lot of advantages overchips that are 1024 ¥ 1024, or larger.

• Lower cost

18 Of two times smaller than the “resolution,” as defined by Rayleigh, or Abbe.19 The array size refers to the number of lines and pixels in the sensor, not to

its total area.

TABLE A3.2. Dynamic Range and Pixel Size

12-bit camera 14-bit cameraw/small pixels w/large pixels

Pixel Size 6.7 ¥ 6.7mm 24 ¥ 24Full Well 27,000 345,000Least significant bit = 6.5 electrons 21 electronsImplied noise level ±13 electrons ±42 electrons


• 4¥ fewer pixels to read out, meaning either:— 4¥ slower readout clock, giving 2¥ lower readout noise.— Same clock speed and noise level but 4¥ faster frame time.

(Easier to scan specimen to find the interesting part! Timeis money!)

• 4¥ less storage space needed to record data.

The argument for big:

• Manufacturing improvements are reducing readout noiselevels at all readout speeds, and CCDs with more pixels oftenalso have smaller pixels which can lead to lower read noise. Ifyour labels are bright, having a larger chip allows you to seemore cells in one image (as long as they are confluent!).Assuming that Nyquist is met in both cases, a large print of animage recorded from a larger sensor always looks sharper thanone from a smaller array.

• Binning: Binning refers to the process of summing the chargefrom neighboring pixels before it is read out. This increasesthe size of each charge packet read (making it look brighter)and reduces the number of pixels. For example: 2 ¥ 2 binningallows the owner of a 1024 ¥ 1024 chip to obtain thespeed/noise performance similar to the smaller chip (512 ¥512) and to do so in a reversible manner. However, the opticalmagnification may need to be increased to preserve Nyquistsampling.

Before deciding that you need a larger chip, compare what youwould get if the same money were spent on anotherscope/CCD/graduate student!

Bottom line:

• If more pixels means smaller pixels, they will each catch fewerphotons unless the magnification is reduced proportionally.More pixels at the same frame20 rate mean somewhat higherread noise because the pixel clock must go faster.

5. Readout Speed:Although readout speed has been discussed above, we haven’tmentioned that some good CCD cameras have variable speed read-outs and the new EM-CCDs impose no high read speed penalty(Table A3.3).

It is convenient to be able to read out the chip faster whensearching and focusing as long as one can then slow things downto obtain a lower read noise in the image that is finally recorded.However, the read speed is only one limitation on the frame rate:

if the signal level is so low that 1 s/frame is required to accumu-late enough signal to be worth reading out, then reducing the readtime much below 0.1 s loses some of its appeal.

Faster readout speeds are particularly important for movingspecimens, especially when doing widefield/deconvolution orwhen following rapid intracellular processes, such as vesicle track-ing or ion fluxes.

6. Shutter Stability:Though not strictly a CCD spec, electronic (LCD) or mechanicalshutters are often built into modern CCD cameras.21 The latter havethe disadvantages of producing vibration and having a limited life-time but the advantage that they transmit all of the light when theyare open (even an “open” LCD can absorb >50% of the light, otherelectronic shutters may be better).

There seems little point in having a camera capable of record-ing (say) 40,000 electrons/pixel with an accuracy of ±200e (or0.5%!) if the shutter opening time is only accurate, or even repro-ducible, to ±10%. If one shutters the light source instead of thecamera, similar limitations apply.

7. User-friendliness:State-of-the-art cameras often seem to have been designed to makesure that no one unwilling to become a devotee of “CCD Opera-tion” can possibly use them efficiently! Start off by asking to seean image on the screen, updated and flat-fielded at the frame-scanrate and showing as “white” on the display screen, a recordedintensity that is only ~5% of the full-well signal. This is where youshould do most living-cell work. Then ask the salesman to helpyou to save time-series of this image. Increase the display con-trast until you see the noise level of the image, both before andafter “flat-fielding.” Put a cursor on one pixel in the top frame ofthe stack and plot its intensity over the series.

8. “The Clincher” (Well, at least sometimes . . .):Ask him/her what the intensity number stored in the computer forsome specific pixel means, in terms of the number of photons thatwere recorded at that location, while the shutter was open. Toanswer, the salesperson will have to know the QE, the fill factorand the conversion factor between the number of electrons in apixel and the number stored in the computer memory (sometimescalled the gain-setting). To help them out, any “real” scientificCCD camera has the latter number written, by hand, in the frontof the certification document (usually a number between 3 and 6).If the salesman doesn’t understand the importance of this funda-mental number, what hope is there for you? (Hint: It is importantbecause the Poisson noise is the sqrt of the number of electrons inthe well, not the sqrt of some arbitrarily proportional numberstored in your computer.)

B. Things That Are (Almost!) Irrelevant WhenChoosing a Charge-Coupled Device for Live-Cell Microscopy

1. Dynamic Range:This is the ratio of the “noise level” to the “full-well” (ormaximum) signal. Although 16-bit may sound a lot better than 12-bit, you need to think before you are impressed.

The noise level should not be more than 5 electrons/measure-ment. Period!

20 The readout speed of a 2 ¥ 2 binned 1024 ¥ 1024 is a bit slower than anactual 512 ¥ 512 because twice as many vertical clock cycles are needed,and one still needs to read out pixel by pixel in the horizontal direction.

TABLE A3.3. CCD Specifications

Array size Pixel Clock Rate Noise level* Frame time Frame rate/s

640 ¥ 480 13MHz 200e/pixel** 0.033 30(video rate)

512 ¥ 512 100kHz 5e/pixel 2.5 sec 0.41MHz 15e/pixel 0.25sec 45MHz 35e/pixel 0.05sec 20

1024 ¥ 1024 100kHz 5e/pixel 10sec 0.11MHz 15e/pixel 1 sec 15MHz 35e/pixel 0.2 sec 5

*Assumes conventional FET circuits. **The readout noise is relatively higher atvideo rate because the higher speed often precludes the use of various techniques,such as correlated double sampling, that reduce readout noise.

21 Often the same advantage can be gained by shuttering the light source. Thismay become more common as pulsed laser or light-emitting-diode lightsources are introduced (see Chapters 5 and 6).


Twelve bits is 4,000 levels. If the first level represents 5 elec-trons (in fact, it should represent half the noise or 2.5e), then the4,000th represents 20,000 electrons or (assuming a QE of 50%),about 40,000 photons/pixel/measurement.

How often do you expect to be able to collect this much signalfrom an area of a living cell only 100 ¥ 100nm in size? You shouldbe able to get a good, 8-bit image using only 6% of the dynamicrange of a 12-bit CCD (Fig. A3.8).22

As the “full-well” signal is only proportional to the area of thepixel on the chip (area in sq. mm ¥ 600), the dynamic range is onlyreally impressive if it is high AND a chip has small pixels. Thenit means that the readout noise is low. A test for actual dynamicrange is described below.

Bottom line: For disk-scanning confocal microscopy, a largedynamic range is only important if it reflects a low readout noiselevel.

Easier to just check the readout noise!

2. High Maximum Signal (high, full-wellnumber, because of large pixels):On living cells, you will probably never have enough light to reacha full-well limit of even 20,000 electrons. Even if you do, thereare better ways to use it (more lower-dose images to show timecourse?).

3. “Imaging Range” “Sensitivity” (or anythingmeasured in LUX):Stick to something you (and I?) understand: Photons/pixel or elec-trons/pixel. The other conversions are not straightforward.

4. “Neat Results”:Unless you know how well stained the specimen is, you cannotevaluate an image of it in a quantitative manner. (Though youmay not want to admit this!) By all means, view your own speci-mens, but viewing “test specimens” that are not expected to fadeand have a known structure (fluorescent beads in some stablemounting medium?) facilitates A/B comparison. If you do use yourown test specimens to compare cameras, be sure to view them onthe same scope, and with the same conditions of pixel size andreadout time etc.

Better still . . .

C. A Test You Can Do Yourself!!!Set up each camera that you want to evaluate on a tripod, add aC-mount lens, and an ND 3 or ND 4 filter. Hook up a monitor orcomputer and view some scene in your laboratory under ordinaryillumination (avoid light from windows which may vary from dayto day).

Close the lens aperture down until you can no longer discernthe image (see Fig. 4.20). This is the “noise-equivalent light level”:the signal level at which the electron signal (i.e., photons/pixel ¥QE) just equals the total noise level. Your measure is the apertureat which the image disappears.23 Because it is sensitive to both QEand readout noise level, this is a very useful measure of what weall think of as the “sensitivity.” Of course, the signal level dependsnot only on the light intensity but also on the exposure time andthe pixel area, so make sure to keep the former constant and makeallowances for the latter.

If you do not have even these meager facilities (a C-mountlens, an ND filter, a tripod and some time), take an image ofnothing. Look at “no light” for one second, and for 100 seconds.Ask to see a short line profile that plots intensity vs. position alonga line short enough that one can see the intensity of each individ-ual pixel. The difference in the average intensity between the shortand long exposure is a measure of the leakage.24 With a little cal-ibration from the published full-well specs (a spec less open to“interpretation” than “noise”), you can even get a direct measureof the read noise level from these dark images. (It should be thestandard deviation of the values as long as they are counted in elec-trons, not “magic computer units” and as long as fixed-patternnoise is not a factor.) And just trying to work it all out will giveyou some idea if the salesman knows anything . . .

D. Intensified Charge-Coupled DevicesIntensified CCDs (ICCDs) are just that: the mating of an “imageintensifier” to a CCD. The idea is that the photon gain of the inten-sifier (can be 200–2000¥) will increase the signal from even a

If the read noise is ±8 electrons, or 2gray levels, one can obtain auseful,“8-bit” (256 levels) image byusing only 6% of its dynamic range.

FIGURE A3.8. Not using the full dynamic range of a CCD. As most scien-tific CCDs have more dynamic range than one “needs” in live-cell fluorescencemicroscopy, the excitation dose to the specimen can be reduced if one sets upthe CCD control program to display an 8-bit image using only the bottom 1,024levels of a 12-bit image. Such an image is more than adequate for many func-tions in live-cell biological microscopy (particularly when other factors suchas dye-loading etc., may cause larger errors) and will require only 6% as muchsignal as would a “full-well” image.

22 Remember, given optical and geometrical losses, you can collect no morethan about 3–10% of the photons produced, and, each fluoroscein moleculewill only produce perhaps 30,000 excitations before “dying.”

23 If the lens doesn’t have a calibrated aperture ring, you can open the apertureall the way and reach the “threshold” exposure level by reducing the expo-sure time and adding ND filters. Remember to also correct for pixel area.Larger pixels intercept more photons.

24 With a good EM-CCD, this measurement can be done using a short exposure and high EM gain, then counting the number of amplified darkcharge/CIC spikes across a typical line of the raster.


single photoelectron above the read noise of the CCD. This occurs,and can be particularly useful where fast readout is needed suchas when measuring ion transients. Finally, pulsing the voltage onthe intensifier section makes it possible to shutter (“gate”) thecamera on the ns time scale, making the ICCD useful for makingfluorescence lifetime measurements (Chapter 27, this volume).

However, ICCDs do not have the photometric accuracy ofnormal CCDs for a number of reasons:

• The relationship between number stored in memory and the number of photons detected is generally unknown and variable.

• The intensifier photocathode has low QE25 (compared to thatof a back-illuminated CCD).

• The “resolution” is generally only dimly related to CCD arraysize because of blooming in the intensifier. To check this,reduce light intensity until you can see the individual flashesproduced by single photoelectrons. See how many lines widethey are. (They should be one line wide.)

• They have additional noise sources: phosphor noise, ions inintensifier section create flashes, high multiplicative noise inthe intensifier section greatly decreases QEeff, etc.

• Photocathode resistivity can produce “dose-rate” effects: non-linearities in which the recorded intensity of the brightest areasmay depend on (and affect) the brightness of nearby features.

Because I expect that EM-CCDs such as those mentionedabove will soon supplant ICCDs except where fast gating isneeded, I have not gone into more detail here. For more info, goto: http://www.stanfordphotonics.com/

ACKNOWLEDGEMENTS

The author would like to thank Dr. J. Janesick, formerly of the JetPropulsion Lab (California Institute of Technology, Pasadena,CA), for many conversations about CCD operation and for theoriginal sketches for Figures A3.1, A3.4, and A3.5 and to ColinCoates, (Andor Technologies, Belfast, UK) for his helpful com-ments on the manuscript and for Figure A3.6.

REFERENCES

Inoue and Spring, 1997, Video Microscopy, Second Edition, Plenum, NewYork, 1-741, particularly Chapters 5–9.

Pawley, J.B., 1994, The sources of noise in three-dimensional microscopical datasets, Three Dimensional Confocal Microscopy: Volume Investigation ofBiological Specimens, (J. Stevens, ed.), Academic Press. New York, 47-94.

25 And the GaAsP photocathode with better QE, have to be cooled, making theassembly very expensive.

Numbers2D imaging, blind deconvolution approach,

476–477.2D-time vs. 3D-time, embryo, 762–764.2D pixel display space, 291.2DCHO dataset, 818.2DHeLa dataset, 818.2-photon, (2PE). See Two-photon excitation.3D Constructor, 282.3D imaging, alternative approaches,

475–476, 607–624. See also,Confocal topics; Multidimensionalmicroscopy topics.

episcopic fluorescence image capture(EFIC), 607–608

light sheet microscopy (SPIM), 613magnetic resonance microscopy (MRM),

618–624amplitude modulation of RF carrier,

620applications, 623–624basic principles, 618–619botanical imaging, 624developmental biology, 624Fourier transform, image formation,

620future developments, 624hardware configuration, 621, 622histology, 623, 624image contrast, 622–623image formation, 619–621Larmor frequency, 620phenotyping, 623schematic, 619strengths/limitations, 622

micro-computerized tomography (Micro-CT), 614–618

contrast/dose, 614–615CT scanning systems, 615–618

dose vs. resolution, 616layout, 614living mouse, 615, 617mouse femur, 616operating principle, 614tumor-bearing mouse image, 617

optical coherence tomography (OCT),609–610

human retina 609schematic, 610Xenopus laevis embryo, 610

objectives on a tandem scanner, 154, 304

optical projection tomography (OPT),610–613

lamprey larva, 612mouse embryo, 612plants, 774–775setup, 611real-time stereo imaging using LLCD

related methods, 607–625selective plane illumination microscopy

(SPIM), 613Medaka heart, 614

surface imaging microscopy (SIM),607–608

3D Scanning Light Macrography, 672.3D for LSM, 282.3D methods compared, 448–451, 644–647.

table, 6473D multi-channel time-lapse imaging

(4D/5D). See also, Time-lapseimaging.

table, 384.3D3T3 high-content screening dataset, 820,

821.3DHeLa high-content screening dataset,

820, 821.3PE. See Three-photon excitation.

4D imaging. See Four dimensional imaging.4Pi microscopy, 561–570.

4Pi-PSF, 570axial resolution, 563I5M, 561, 569–570

OTF, 569–570living mammalian cell imaging, 564–565

Golgi apparatus, image, 566lobe-suppression techniques, 561

interference of excitation and detection,561

confocal detection, 561two-photon excitation (2PE), 561

MMM-4Pi microscopy, 554, 556,563–564

basics, 565scheme, 563

optical transfer function (OTF), 562, 563outlook, 568–569point spread function (PSF), 562–563signal-to-noise ratio, 561space invariance of PSF, 457, 490, 564theoretical background, 562–563type C, with Leica TCS, 4Pi, 565–568

imaging of living cells, 568lateral scanning, 567mitochondrial network, image, 568optical transfer function (OTF), 567resolution, 567sketch, 566thermal fluctuations minimized, 567

z-response, 5635D image space, display, 291–294.

2D pixel display space, 291animations, 292–293color display space, 291efficient use, 292image/view display options overview,

table, 293

Index

933

I suppose it is inevitable that indexes are compromises: If one includes every mention of every entry, the index becomes as long as thebook. There is also the time dimension: As one cannot start writing the index until the book has been paginated, every day spent onthe index directly delays the publication date. For the Second Edition, I prepared the index somewhat in parallel with the page proofsand it took most of a semester. For this Third Edition, a professional indexer was used to compile the initial index. We then expandedthe level of cross-referencing through a series of digital searches. The final result may show its mixed parentage.

As you use this index, please consider the following. I confess that many entries contain far fewer referents when they appear assub- or sub-sub-heads than when they appear as capitalized headings. In addition, some See alsomarkers use acronyms and it is alsotrue that these can get confused with the real title of the entry. In compensation, have tried to put in bold type those page numbers on which I one would find the more comprehensive discussions of the topic we have added a period at the end of the major heads todistinguish them from sub-heads. My apologies for any errors.

My thanks to Helen Noeldner for her calm and competent assistance during this long and laborious process. Please use the Feed-back page at http://www.springer.com/387-25921-X to bring errors to our attention so that they can be corrected in future printings.Remember that this Handbook has always been a community project. Good hunting!

JP, 2/21/06

934 Index

5D image space, display (cont.)multiple channel color display, 292optimal use, 293–294pseudo-color, 173–175, 190, 291stereoscopic display, 293true color, 291

AAbbe, Ernst, 1, 5.Abbe refractometer, 377.Abbe resolution criterion, 36, 37, 60, 61,

65–68, 574, 575, 631–636, 928. Seealso, Rayleigh criterion.

breaking the Abbe limit, 573calculation, 65–66individual point features separated by,

68pixel size, 62, 65, 634–635, 784, 928

Abbe sine condition, 151, 239.Abbreviations, list, 125.Aberrations, 109, 146–156, 241, 411–412,

471, 480–481, 542, 629, 640–641,654, 655, 657–659, 747. See also,Chromatic aberrations; Refractiveindex mismatch; Sphericalaberration.

astigmatism, 145, 151–152, 245–247,249, 483,

axial, 242, 505, 542, 630chromatic, 152–156, 160, 177–178, 209,

242–243, 641, 659in 2-photon disk scanning, 542, 550,

554of AODs, 56axial chromatic registration, 287, 658chromatic registration, 657–658of collector lenses, 657–658intentional, for height measurement,

224magnification error, 155, 287, 331, 493,

542, 641, 657, 883, 904measurement of, 243–244, 654, 659multi-photon microscopy, 542of optical fibers, 504, 507signal loss, in confocal, 156, 178, 542,

641standards, table, 157

coma, 145, 151–152, 245–246, 249, 483,630

detecting, 241monochromatic, 147–152, 542. See also,

Spherical aberrationoptical, avoiding with thin disk lasers,

109of refractive systems, 146–156signal loss, 156, 178, 542, 641spherical, 15, 34, 147–149, 151, 160, 192,

208, 241, 244, 247, 330, 395,404–413, 454–455, 463, 466, 480,542, 629, 640, 654–655, 657, 658,728, 772, 774. See also, Sphericalaberration; Mismatch, refractiveindex

blind deconvolution to remove,480–481

cause of signal loss, 330, 389, 395,413, 457, 542, 661

chapter, 404–413correction for refractive index

mismatch, 192, 287, 411–412, 542corrections for, 145, 411–412, 654–655corrector optics, 192, 395, 398, 411,

477, 640, 655, 657deconvolution, 463, 466, 468, 469, 471,

480, 498–499, 784generated by specimen, 192, 418,

454–455, 654, 658, 747, 772, 775of GRIN lens, 108for IR wavelengths, 160measurement using small pinholes,

145, 407monochromatic, 147–151multi-photon excitation, 542, 407–410PSF, 148, 407, 455, 471, 481, 492, 657secondary, 247, 249in thick embryo imaging, 747Zernicke coefficients, 247, 248

wave-front, measuring performance, 145Ablation, 2-photon, 107, 764–765.Absorber, saturable-crystal, 107, 111, 112.

to cover gap in titanium:sapphire lasers,112

indium-gallium arsenide, InGaAs, 111Absorption, 25, 163, 309–312, 338–339,

341, 514–518, 542, 550, 613, 704.2-photon, 405, 535–536, 541, 545, 550,

552, 705, 719, 764, 884caged compounds, 543, 544CARS, 595–596, 599contrast, 162–165, 211, 595, 610, 613,

770, 779cross-section, 189, 426energy levels, 514, 517, 682, 697, 705,

792excited state, 544, 692fiber optics, 501, 502filters, 552of fluorescent dyes, table, 345fluorescent excitation, 45, 88FRET, 184and heating, 21, 218, 252, 539, 685of incident light, 163, 177, 427by ink, 73and laser operation, 82, 108, 110, 116light lost by, 25, 166, 414–418, 457, 654lighting models, 283, 285, 309–312molar extinction, 80–81, 343, 353, 357,

793nonlinear, 188, 416, 427, 680, 704,

709–710of optical materials, 158and photodamage, 22, 685–686, 690, 750in photodetectors, 253photon, 550, 749quantum dots, 221, 343, 357–358, 696,

759, 801

RESOLFT/STED, 573self-absorption, 490spectra, 217, 267, 338–339, 345, 355,

390, 415–416, 421, 538–539,681–682, 706

in UV, 195Absorption coefficient, complex specimen,

164.Absorption contrast, 164–167, 195, 427.

equations, 164, 539heating, 539, 685

Accuracy.biological vs. statistical, 24, 36–37, 68,

312position, 39–41

Acetoxymethyl ester indicators, 726.deposits formaldehyde, 738derivatization, 738formula/reaction, 359, 738loading method (AM ester), 358–359,

361, 726, 738–739, 744painting brain slices with, 726–737

Achrogate beam-splitter/scan mirror, 50,212, 231–232, 916.

operation, 50, 232, 916Zeiss LSM5 line scanner, 212, 231–232,

916Achromat, 152, 153, 244.

chromatic correction of, 153flatness of field and astigmatism, 152longitudinal chromatic correction, 153measurement, 244

Acousto-optical beam splitters (AOBS), 45,55–57, 88, 102, 211, 218, 395.

to select wavelength and intensity, 88,102

to separate illumination and emission, 45,218

Acousto-optical components, 43, 54–57.tellurium oxide crystal, 55thermal stability, 56, 57, 219

Acousto-optical deflectors (AOD), 25, 33,54–56, 88, 447, 519, 543, 664, 762,908.

as beam-splitters, to reduce loss, 33to gate light source, 25. See also, AOM

group velocity dispersion due to, 88,540, 646

multi-photon excitation, 88, 540, 543, 646

multi-tracking, 664performance, 55problem descanning fluorescent light, 56,

447Acousto-optical modulators (AOM), 11,

55–57, 88, 231, 519, 540, 543.FRAP experiments, for controlling laser,

56group velocity dispersion, 88

Acousto-optical tunable filters (AOTF), 43,55–56, 88, 102, 219, 237, 346, 543,651, 660, 673, 806, 908.

for selecting CW laser lines, 88, 102blanking, 54, 55, 237, 389, 543, 628, 651

Index 935

leakage, 660to regulate light intensity, 43to spectrally filter light, 55thermal sensitivity, 56–57, 219

Acridine Orange, 23, 344, 531, 665–667,691, 774, 874.

bleaching, 693–694Acronyms, list, 125.Actin filament, 7, 236, 372, 378, 383, 692,

696, 714, 719, 748–749, 753, 756,759–760, 773, 781, 804, 819,824–825, 854, 856.

widefield source suitability, 142Active laser medium, defined, 81.Active mode-locked, pulsed laser, 111.Actual focal position (AFP) defined, 405.Actuator, galvanometer, 52.Acute neocortical slice protocol, 723.Adams, Ansel, zone system, 71–72.Adaptive optics, 892.ADC. See Analog-to-digital converter.Adipocyte cells, CARS imaging, 604.Adjacent fields, automated confocal

imaging, 810.ADU, analog digital units, 74–77, 630, 925.Advanced Visual System. See AVS.Aequorea victoria, biofilms, 348, 356, 736,

794, 873–874, 877.variants, table, 873, 874

Aequorin, Ca2+ reporter,736–737, 739, 741,802.

developmental cellular application, 736ion binding triggers light emission, 737Ca++ signal detection, 737

AFP. See Actual focal position.AIC. See Akaike Information Criterion.Airy aperture, optimum for NA, 28.Airy disks, 4, 24, 65, 131, 145–146, 151,

156, 210, 443, 444–449, 454–456,463–465, 474, 485, 492–493, 562,567, 630, 655–657.

Abbe criterion resolution, 65–66, 225defined, 146, 444diameter in image plane, 210, 225four-lobed, from astigmatism, 151image, 38, 146, 225intensity ratios, 28, 145–146inverse, 11and line spacing, 24radius and pixel size, 4, 24, 38, 39, 60,

65–67, 227, 485vs. NA and wavelength, 1, 4, 146

Airy figure image, 38, 75, 79, 146, 147,225, 479, 486–487, 562.

FWHM as optimal pinhole/slit size, 28,36, 225, 232, 443, 454, 463–465,564, 567–568, 630–631, 633,655–657

and resolution, 65–67size, and Nyquist criterion, 38, 39, 60

Airy unit, 28, 36, 41, 210, 222, 227, 232,274, 443–451, 632, 775, 779.

Akaike Information Criterion (AIC), 825.

AlexaFluor dyes, 81, 103, 184–185, 190,192, 236, 330, 342–344, 353–357,360, 363, 393, 395, 416, 533, 540,694, 726, 731, 749, 794, 799, 804,810, 814, 854, 878, 880, 905.

fluorescence excitation, 355living cells rapid assessment, table,

360structure, 356

Alexandrite (Cr3+ in BeAl2O4), tunable laser,109.

Alga.autofluorescence, 357autofluorescent image, 173, 175, 192,

194–195, 438–439, 528, 585, 785,870, 881–885

biofilm, 870, 881–885cell chamber for, 429in laser cooling water, 116

Aliasing, 38–39, 271, 291, 293, 448, 588,590–592, 640, 830, 833–834,836–839, 903.

and Nyquist criterion, 38–39, 448temporal, 39, 41, 391, 836–837, 839

Alignment, 25, 85, 134–135, 157, 505,629–631, 651.

of laser systems, to reduce instability, 85

of optical coherence tomography, 610of optical system, thermal stress, 85

importance, 25, 630and PSF, 646of source, 134–135, 629–631

Alkali vapor lasers, diode-pumped,103–105.

Allium cepa. See also, Onion epithelium.Alpha blending, 302, 304.Alumina (Al2O3) ceramic tubes for lasers,

102.Amira, 282–283, 286, 296, 302, 308, 312,

775–778.Amoeba pseudopod, detail, 168.Amplifier rods, maintenance, 116.Analog digitization, for photon counting, 29,

33–37, 41, 65, 74, 78, 251, 254,258–261, 263–264, 404, 460, 495,522, 525–526, 542, 634, 766.

Analog-digital unit (ADU), to calibrateCCDs, 74, 77, 630, 925.

Analog-to-digital converter (ADC), 31–34,64–66, 70, 72, 74–75, 258–259, 261,263, 286, 521, 630–632, 924–925.

Analyze (software), 281–282, 288, 290,301–304, 312, 651.

Analyzer, in pol-microscopy, 25, 157, 229.Analyzer, spectrum, 901–902.Anemonea majano, sulcata, 874.Angular deflection, distortion, 211.Aniline Blue stain, 430–432, 435, 438, 774.Animations, 281, 283–285, 289–290,

292–293, 295, 299, 308, 312, 764,829, 835–839, 841–844.

Anisotropic crystals, 114.

Anisotropic sampling, 287–288.when resampling, 833–835

Anisotropic specimens, 163, 286, 320, 329,420, 623, 675, 678, 690, 710, 793.

Anisotropy analysis, chimeric proteins, 794.Anisotropy of fluorescence, 742, 794.Anisotropy of interference filters, 49.Annular aperture, 4, 9, 20, 211, 889.

3D pattern of point-source from lens,4–20

in specimen-scanning confocalmicroscope, 9

Anti-bleaching agents, 36, 340, 363, 368,375, 499, 694.

Antibody stains, 292, 339, 342–343, 348,357–360, 375, 528, 576–578, 582,610, 612, 664, 696, 731, 748, 760,789, 802–804, 812, 852–855,877–880.

artifacts, 664biofilms, 877–880FRET, 790–791high-content screening, 812–815, 818in situ, 612penetration, 387preparation, 369, 371–372, 375–377, 878and TEM, 852–855

Antifade agent, 36, 340, 363, 368, 375, 499,694. See also, Antioxidants.

Antiflex optics, to reduce reflections, 158,171, 507, 513.

Antioxidants, living cell imaging, 341–342,363, 389, 390, 729, 794.

Anti-reflection (AR) coatings, 1, 8–9, 25,49, 117, 139, 145, 151, 158–159,212, 505–506, 901.

color effect, 139of optical fibers, 506

AOBS. See Acousto-optic beam-splitter.AOTF. See Acousto-optic tuning filter.APD. See Avalanche photodiode.Apochromat, 15, 147–148, 151, 153–155,

158, 240–245, 409–410, 454–455,655, 659, 771.

chromatic correction, 153compared with fluorite objective, 154longitudinal chromatic correction, 153

Apodization, high-NA objective lenses, 240,243, 249–250, 272, 567, 889.

Applied Precision Instruments (API), 131,137, 282, 388, 651.

APSS up-converting dye, saturation, 165.AR. See Anti-reflection.A. thaliana, 169, 173, 174, 175, 193, 196,

202, 416, 420–421, 423, 425, 426,427, 431, 771, 772, 773, 775, 778,779, 780.

attenuation spectra, 416birefringent structures in cells, 420–421.

See also, Anisotropic specimensbleaching, 203

double imaging, 169fluorescence spectra, 421, 423, 425

936 Index

A. thaliana (cont.)GFP protein fusion, 773limitations for imaging, 772mesophyll protoplasts, 196, 426optical sectioning, 772, 775protoplasts, 195–196, 203, 416, 421,

425–427, 429–430 438–439, 693root tip fluorescence spectra, 173–175seedling, autofluorescent image, 202three-dimensional reconstruction, 190,

193, 771, 775, 777–778, 781two-channel confocal images, 169, 175,

193, 196, 203, 427, 431, 772two-photon excitation, advantages, 779,

780two-photon fluorescence image, 427, 780two-photon fluorescence spectra, 425, 426

Arc lamps, 132, 136–138.current/stability of plasma, 138–139monitoring during exposure, 137radiance, 137–138sensitivity to environmental variation, 136shape of discharge, 132shift of wavelength with temperature, 137stability of, vs. filament lamps, 137

Area of interest. See also, Region ofinterest.

identifying, 201–202Argon-ion laser, 85–86, 90–102, 107,

109–110, 112, 119, 124, 203, 338,341, 346, 353, 355, 375, 540–541,655, 657.

CW, 90–103, 107, 109–110, 112, 119, 124

emission stability, 86, 102references, 124

Argon-krypton mixed-gas laser, 90, 92, 93,102, 108, 119, 203, 343, 375, 748,798, 811.

Artificial contrast, vibration and ambientlight, 201–204.

Artificial lighting, image display, 306–312Astigmatism, 145, 151–152, 245, 247, 249,

483, 505, 542, 630.of AOD, 914and flatness of field, 152and intensity distribution, 152, 246, 630laser optics, 89, 106–107, 505measuring subresolution pinholes, 145at off-axis points, 151, 245, 247, 249

ATP-binding cassette, 362.ATP-buffer, 802–803, 812.ATP-caged, 544.ATP-gated cation channels, 359.Attenuation of light.

by specimen, 164, 287, 298, 304, 320,321, 414–418, 428, 439, 538, 558,706, 779, 782

plots, 415, 706of laser beams, 85, 87, 354, 415, 904modeling, 309, 311, 320–321, 330of PSF, 456, 462–463, 466, 494x-ray, 614–615

Atto Bioscience CARV confocalmicroscope, 215, 229, 230, 907.

Autofluorescence, 44, 81, 90, 173, 175, 195,202, 339–340, 360–361, 369–370,387, 414, 416, 421–434, 442–445,447–449, 451, 509–510, 528, 530,545, 607, 612, 663, 667–670, 678,682, 690, 698, 706, 710–711, 713,729, 742–743, 745, 764–765,769–773, 779, 781–782, 785, 798,815, 874, 876, 881–885.

of alga chloroplast, 168, 172–176, 202,429–435, 556, 785

A. thaliana seedling, 202, 303, 307, 772

bleaching, 202, 698, 729cell wall, 303, 431, 438, 770emission spectra in plants, 176, 421–423extracellular matrix, 311fixation, as a cause, 358, 369–370fluorescent probes, 339–340, 360–361harmonic signals. See Harmonic signalslamprey larvae, 612multi-photon microscopy (MPM) See

also, harmonic signals, 545optical materials, 45, 158plants, 190, 193–195, 421–428, 770–772plots, 176, 421–423removal using spectral unmixing, 192,

382, 664–667examples, 665–666

removal on basis of fluorescence lifetime,345–346, 348, 349, 528

UV excitation, 347Automated 3D image analysis methods,

316–335. See also, Automatedinterpretation of subcellular patterns.

biological objects, 319blob segmentation example, 322–324

gradient-weighted distance transform,323

model-based object merging, 323–325watershed algorithm, 322–325, 777,

822combined blob/tube segmentation,

328–330data collection guidelines, 319–320defined, 316, 328future directions, 334hypothesis testing, 318illustrations, 317image preprocessing, 320–321

background subtraction, 320morphological filters, 320signal attenuation-correction, 320–321

vs. manual, 316–317montage synthesis, 282, 293, 312,

328–332, 748, 753, 851–852, 855,858–859

defined, 329–330examples, 330–332, 780–781neuron, 330

scanning electron micrographs,851–852, 855

TEM implementation, 858–859neurobiology example, 320quantitative morphometry, 331rationale, 316registration synthesis, 328–331

defined, 328landmark-based, 328–329multi-view deconvolution, 291, 330,

675–677segmentation methods, 321–322

bottom-up, 321hybrid, bottom-up/top-down, 322integrated, 322intensity threshold-based, 321region-based, 321–322top-down, 322

segmentation testing methods, 333–334manual editing, 333–334

specimen preparation, 319–321imaging artifacts, 320

stereology, 316time series in vivo images, 319tube-like object segmentation example,

324–328mean/median template response, 328skeletonization methods, 324–325vectorization methods, 324, 326,

327types, 318–319

Automated fluorescence imaging, 814.endpoint translocation assays, 814

Automated interpretation of subcellularpatterns, 818–828. See also,Automated 3D image analysismethods 2D dataset analysis.

automated 2D analysis methods, 8182D subcellular location features,

819–8202DHeLa dataset images, 819CHO cell dataset, 818, table, 820Haralick features, 818–820HeLa cells 2DHeLa dataset, 818Zernike moments, 818–820

automated 3D analysis methods, 824classification results, 824feature normalization, 824feature selection, 824

automated classification of locationpatterns, 824–825

classification accuracy, 826confusion matrix for 3DHeLa images

using SLF10, table, 824confusion matrix for 3DHeLa images

using SLF17, table, 825features in SLF17, table, 825measured classification accuracy, table,

825clustering of location patterns with

clustering consistency, table, 826exclusion of outliers, 825methods, 826

Index 937

optimal clustering determination,825–826

optimal consensus tree, 827clustering of location patterns, 825–826downsampled images, different gray

scales, 824–825future directions, 827–828high-resolution 3D datasets, 820–822

3D3T3, 8203DHeLa, 820color images from 3DHeLa, 821image acquisition requirements,

821–822images from 3D3T3, 821

image database systems, 827image processing/analysis, 822–823

3D SLF, 822–823edge features, 823feature calculation process, 822morphological features, 823segmentation of multi-cell images, 822texture features, 823

protein subcellular location, 818statistical comparison of patterns,

826–827AutoMontage software, 282, 293, 304.Avalanche photodiode (APD), 77, 233,

252–255, 404, 527, 542, 558, 567,698.

array, for multi-beam sensing, 558noise currents, 256pulse pileup, 253, 527unsuitability for non-descanned detection,

542vacuum ADP, 254–255

Average intensity, 66, 110, 516, 556, 668,684, 695, 747, 763–764, 816, 838,930.

equation, 302, 309, 668AVS (Advanced Visual System), 282–283,

286, 300, 311–311, 862, 863.Axial chromatic aberration, 155, 658–659.Axial chromatic registration, 154, 658.Axial contrast. See z-contrast.Axial edge response, 409–410, 654.

calculations for glycerol, table, 409calculations for water, table, 409

Axial illumination, 60–61, 134.Axial laser modes, 82, 110.Axial minimum, 3D diffraction pattern, 4,

147.Axial rays, spherical aberration, 148.Axial resolution, 3–4, 6, 172, 182, 209, 211,

225–228, 230, 240–241, 243–244,320, 370, 395, 407–411, 413,444–446, 489, 493, 499, 511, 513,551–553, 559, 561–568, 571–577,610–611, 613, 649, 651, 654,656–657, 659, 674, 704, 747,750–751, 822.

4Pi microscopy, 561–568coding, display, 305defined, 3–4, 240, 444–446

focus shift, 243, 407–410as function of pinhole diameter, 656magnification, 215measurement, 194, 656–657, 659multi-photon, 750multiview, 678near focal plane, slit-/point-scan confocal

microscopes, 225–228SHG, 704SPIM, 614, 674, 751STED, 571–577tandem-scanning confocal microscope, 6,

225tomography, 610–611using mirror, 656–657

BBack-focal plane (BFP), 34, 50–51, 58,

61–62, 84, 126–128, 166, 208–210,225, 239, 268, 487, 509, 627, 629,708.

Background light, from transmissionilluminator, 201–202.

Background noise, 260–262, 275.Background signal, 12, 26, 28, 37, 68–69,

71–72, 88, 90, 112, 115, 158, 162,168, 172–173, 175, 184, 188,201–202, 221–225, 227, 232, 235,248, 251, 257, 266–275, 278–279,283, 287,-288, 290, 301–302, 305,312, 321, 326, 339–340, 343, 345,348, 360–362, 375, 421, 423,428–429, 432–433, 442–451, 462,465, 472–477, 486, 493, 497, 506,509–510, 518–519, 535, 541, 543,553, 559, 582, 584–585, 595,598–600, 602, 604, 621, 633, 656,663–370, 676, 694, 697, 698, 707,713, 727, 733–734, 736, 747,755–757, 760, 798, 801, 803, 809,813, 815, 818, 822, 830, 836, 839,851.

Background subtraction, 284, 301, 320, 473,510.

Back-illuminated CCD, 31, 77, 222, 232,234, 754.

Back-propagation neural network (BPNN),818.

Backscattered light (BSL), 22–23, 57,83–84, 130, 141, 145, 165, 169–170,180–182, 191, 196, 202, 212, 221,228, 240, 376, 378, 416, 430, 436,442, 631, 879.

access to, antiflex optics, 6, 57, 141, 212,229, 507, 513, 609, 631, 704, 707,854, 879, 990

biofilm, image, 880contrast, effect of specimen absorption,

165effect of coherence on, 130–131, 170images made using, 22–23, 154, 436–438,

513, 638, 855, 880Amoeba pseudopod, 168–170, 191

cheek cells, 22, 23diatom, 145, 438, 638–640, 881latex bead, 182, 196, 197, 653transparent ciliate protozoa, 141

LLLCD objectives/3D color-coded BSLas a noise signal, 663optical coherence tomography, 609practical confocal microscopy, 631from specimen, 202unmixing, 192, 382, 664–667

Back-thinned CCD, 31, 77, 222, 232, 234,754.

QE plot, 29Bacteria. See Biofilms.Ballistic microprojectile delivery, 360, 726,

803.Ballistic photons, 418, 427, 538.Ballistic scans, 40, 41.Balloon model segmentation methods, 776.Bandpass, optical filters, 43–44, 46, 48, 49,

51, 76, 87, 132, 141, 173, 204, 341,528, 708, 798.

for CARS, 598–599coupling short and long-pass filters, 46excitation and emission, 48, 141, 217,

341, 708, 757, 798laser, 106–107liquid crystal, 425to select range of wavelengths, 43–44spectral detector, 203–204, 662–663,

666–667Bandwidth, 32, 64, 69.

3dB point, definition, 59, 65of AOBS, 57electronic/optical, digitization, 32, 34, 70,

238head amplifier, 251limiting, to improve reconstruction, 69Nyquist reconstruction, output, 64, 69, 70,

238Bead, fluorescence emission, 181, 182, 196.

fluorescent, 454, 477, 493, 499, 527, 652,653, 656, 659, 784, 900, 904, 930

image, 656table, 653

glass, in water, 181, 198–199latex, fluorescence image, 196, 407,

455–457, 463, 471, 656in water, confocal serial sections, 182

Beam blanking, 54, 55, 237, 389, 543, 628,651.

Beam collimation, 728.for fiber delivery, 506

Beam delivery, with fiber optic coupling,85–88, 107, 216, 503, 506–508.

Beam deviation, unintentional, 15–16.Beam expander, 8, 84, 124, 208, 212–214,

231, 650, 682, 708, 728, 907.advantages, 213

Beam pointing, lasers, 85, 103, 107, 201,250.

active cavity stabilization, 87Beam quality, of diode lasers, 107.

938 Index

Beam shift, vignetting due to, 211.Beam-splitter, 33, 46–48, 50–51. See

Dichroic mirrors.Achrogate, 50, 212, 231–232, 916AOBS, 56–57broadband, 346dichroic, 25, 33, 35, 43–51, 56–57,

83–84, 88, 139, 132, 135, 143, 151,162, 203–204, 207–208, 211–214,217–218, 229, 231–232, 266, 339,341, 346, 375, 386, 424, 469,503–504, 552, 563–564, 599,630–632, 647, 650, 657–658, 664,667, 691, 707–708, 747, 771–772,810, 846, 879, 910, 907

table, 799fiber-optic, 503–504forty-five degree, performance, 47fused-biconic coupler, 503–504long-pass cut-off, 43, 46, 51, 175, 204,

564, 801, 875multi-photon, 540–541polarizing, 13, 50, 57, 85, 87, 100, 217,

513, 631spectral problems, 50–51triple dichroic, 33, 46, 48, 217–218, 658,

783losses due to, 33performance, 46–48

Beam scanning, along optical axis, 215,555.

Beam-scanning confocal microscope. SeeConfocal entries; Flying spotultraviolet (UV) microscope.

chromatic correction, 177Beam-scanning systems, 6, 7, 16, 132, 146,

151, 156, 166, 177, 214–215, 218,381, 554, 562, 564, 567, 568, 599.

coma in, 151off-axis aberrations affecting, 156

Before-bleach/after-bleach ratio, FRET, 794.Benchtop fiber-optic scanning confocal

microscopes, 507–508.Bertrand lens, 61, 157, 412, 643.Beryllium oxide (BeO), for laser tubes, 102.Beta barium borate (BBO), non-linear

crystal for frequency doubling, 100,109, 114–115, 125.

BFP. See Back-focal plane.Bibliography, annotated, 889–899.

adaptive optics, 892books on 3D light microscopy, 889differential phase contrast, 892display methods, 892–883fiber-optic confocal microscopes, 883general interests, 891historical interests, 889–890index mismatch, 893–894multiplex, 894non-linear, 894point spread function, 895–896polarization, 894–895profilometry, 895

pupil engineering, 896review articles, 889technical interests, 891–892theory, 890–891thickness, 896turbidity, 896–897variants on main theme, 897–899

Binding equation, for fluorescent indicators,740.

Biocytin, 730, 731.EM imaging of brain cells labeled, 731protocol, 730

Biofilms, 287, 688, 529, 530, 624, 779,870–887.

2-photon imaging, 530, 882–885dual-channel imaging, 884limitations of CLSM and 2-photon, 884single-photon/2 photon comparison,

883thick environmental biofilms image,

885autofluorescence, 545backscattered light, 880fluorescent proteins for, table, 874future directions, 887GFP variants for, table, 873imaging extracellular polymeric

substances (EPS), 879–882lectin-binding analysis, figures, 881,

882lifetime imaging, 530magnetic resonance microscopy, 624making bacteria fluorescent, 873–874pH imaging, 530, 739–745sample mounting, 870–873

flow chamber system setup, 872–873perfusion chambers, 870–872pump selection, 871upright vs. inverted microscopes, 870,

872water-immersible lenses 149. 161, 209,

411, 429, 568, 613, 727, 737, 870,872.

stains for, 874–879, 875Acridine Orange, 23, 344, 531,

665–667, 691, 774, 874antibodies, 877–878biofilm community on tooth, 879DAPI, 874. See also, DAPIeffect of antibiotic treatment, 877embedding for FISH, 876–877FISH with fluorescent protein,

875–876, 878imaging bacteria, backscattered light,

879live/dead stain, Streptococcus gordonii,

876nucleic acid, 874–875preparing labeled primary antibodies,

878SYTO, 874–875

temporal experiments, 885–886multi-cellular biofilm structures, 886

time-lapse confocal imaging, 885–886transmitted laser light image, 880

Bioimagers, kinetics, endpoint analysis,816–817.

Biolistic transfection, 360, 724–726, 803.Biological accuracy, vs. statistical accuracy,

24, 36–37, 68, 73, 312.Biological reliability, of measurements, 24,

36–37, 68, 73, 312.Biological specimens, 6, 11, 12–13. See

also, Plant cell imaging, Biofilms,Specimen preparation, and entriesunder specific equipment andcell/tissue type.

backscattered light images, 22–23, 25,167–168, 170, 880

CARS imaging, 603–604adipocyte cells, 604epithelial cells, 603erythrocyte ghosts, 603

distortions caused refractive indexinhomogeneity, 40–41, 181, 182,

198–199, 419tandem scanning systems for, 6, 11Yokogawa CU-10, 12–13

Biophotonic crystals, 188, 428.Bio-Rad, 25, 33, 35–36, 70, 113, 214, 260,

630, 638–640, 657, 748–752, 757,759–762, 858, 889.

1024ES, 710–711, 714, 718–719data storage, 585using white light source, 113

MRC 1024, photon counting, 33photon efficiency, 25, 32, 261,

748–752MRC-600 scanner, full-integration

digitizer, 70PMT, 260–261Radiance-2100, 23, 185resolution, 657

Biosensors, fluorescent, 33–8348, 799, 805.See also, Dyes, Fluorophores, andChapters 16 and 17.

future, 805mitotic clock measurements, 799

Birefringence, 6, 15, 54, 83, 103, 109, 113,116, 162–164, 188, 189, 414,420–421, 431, 434, 436, 438, 479,503, 710–711, 714, 717, 894.

acousto-optics, 54, 55collagen fibers, 164, 188, 717contrast, 15, 162–164, 188, 414–428,

431–438, 710–711, 714, 717, 719,894

deconvolution, 479–480defined, 163, 188in fiber-optics, 503harmonic generation from, 428, 431–438images of Cymbopetalum baillonii, 189in laser components, 85, 103, 109, 113,

116quarter-waveplate, 6table, 715

Index 939

Birefringent crystals, 188, 420–421.optical effects of acoustic fields on, 54,

55Black-body radiation, 44, 135–136.

from incandescent lamps, 44, 126,135–136

spectrum, 136Bleaching, 10, 12–13, 20, 24, 44, 63–64, 90,

142, 186–187, 194, 202–203, 210,218, 220, 222, 340, 382–387, 442,539–540, 690–702, 797, 905, 907.

2-photon excitation, 539–540, 680–689,905

acceleration, 341of acceptor in FRET, 184–187anti-bleaching agents, 36. See also, Anti-

bleaching agentsbleach patterns, 3D, 538, 628, 693beam blanking, to reduce, 53–54before/after ratio, for donor/acceptor pair,

794chapter, 690–702combining fluorescence with other,

383–386in dye lasers, 103dynamics, 202–203fluorescence correlation spectroscopy,

383, 801fluorescence lifetime, 382–383fluorescence recovery after

photobleaching, 51, 54, 56, 80, 90,187, 210, 218, 224, 229, 237, 362,382, 684, 390, 691, 759, 801, 805,850

FRET, 186, 382, 794–798, 800fluorescence speckle microscopy, 383in four-dimensional imaging, 222improvement, recent, 36laser trapping, 383linear unmixing, 192, 382, 664–667of living cells, 212, 220, 382, 797. See

also, FRAP, FLIPintensity dependence, 341, 363mechanism, 222–223of non-specific fluorescence, 27, 44, 74optical tweezers, 383, 385performance limitations, 221, 224, 232,

381, 448–450, 556, 693. See Chapter39

photoactivation, 187, 224, 383, 385, 541,544–545, 693, 759

photo-uncaging, 383. See also, Photo-uncaging and signal per pixel, 63–64

spectral unmixing, 192, 382, 664–667table, 384–385techniques, 125temperature as a variable, 696–698time-lapse fluorescence, 382

Bleedthrough fluorescence, 185, 203, 664,904.

multi-tracking, reduces bleed-through,664

Blind deconvolution, 190, 468–487. Seealso, Deconvolution.

2D approach, 476–4773D approach, 475–476advantages/limitations, 468–472algorithms, 472–474of A. thaliana seedling image, 190confocal stack, 470data collection model, 472data corrections, 477defined, 469DIC schematic, 475DIC stack example, 470different approaches, 475–477

deblurring algorithm, 476Gold’s ratio method, 476inverse filter algorithm, 476iterative constrained algorithms,

475–476Jansson-van Cittert algorithm, 476nearest-neighbor algorithm, 476no-neighbor algorithm, 476–477processing times/memory table, 476Richardson-Lucy, 497, 568TIRF microscopy, 477

differential interference contrast (DIC),473–475

examples, 469, 470, 481, 482, 483flowcharts, 473, 474future directions, 483Gerchberg-Saxton approach, 472hourglass widefield PSF, 474light source/optics alignment, 478maximum likelihood estimation (MLE),

472–477, 669–670new developments, 478–480

live imaging, 480polarized light microscopy, 479subpixel imaging, 478–479

optical sectioning schematic, 469OTF frequency band, 474simulated example, 481, 482speed, 482–483spherical aberration correction, 480–481,

471spinning-disk confocal example, 481,

482, 482transmitted light, bright-field (TLB), 472,

477two photon example, 481, 483widefield simulated example, 481, 469WWF stack example, 469

Blind spots, due to sampling with largepixels, 38.

Blue Sky Research, ChromaLase 488, 107.Boar sperm cells, 557.BODIPY dye, 142, 342–343, 353–356, 389,

692, 749, 760–762.BODIPY TR, methyl ester dyes, 760–762.Bolus injection protocol, 360, 726, 728,

731.Bone, reflectance, 167.Books on 3D LM, listing, 889.

Borohydride, to reduce glutaraldehydeautofluorescence, 374, 770.

Botanical specimens, 414–439, 624,784–785. See also, Plant cellimaging, and Chapters 21 and 44.

birefringent structures, 420–421. See also,Birefringence

deconvolution, 784–785effect of fixation on, 195, 428Equisetum, 774fluorescence properties, 421–428

emission spectra, 421–423microspectroscopy, 421–426

fluorescence resonance energy transfer,425.

See FRET, 425harmonic generation properties, 428,

711–715light attenuation in plant tissue, 414–418

absorption spectrum, 415A. thaliana example, 416maize stem attenuation spectra, 417,

418M. quadrifolia attenuation spectra, 416M. quadrifolia optical sections, 419Mie scattering, 162–163, 167, 417–418nonlinear absorption in, 416–417Rayleigh scattering, 162–163, 167, 417,

703light-specimen interaction, 425–428living plant cell, 429–439

calcofluor staining procedure, 424, 438callus, 429cell walls, 168–169, 188–189, 303,

306, 416–417, 420–421, 428–431,435–136, 438, 439, 710–711,713–715, 769–776, 779–781

chamber slides, use, 429culture chamber, 429cuticle, 434–437, 715, 717, 779fungi, 438–439, 624, 782, 870hairs, 431, 434–436, 772meristem, 168, 420, 430, 770, 776–778,

783microsporogenesis, 431–432mineral deposits, 163, 420, 436–438,

703pollen germination, 420, 433–434, 781,

783pollen grains, 202, 305, 313, 420,

431–433, 553, 558, 781, 783protoplasts, 195–196, 203, 416, 421,

423–427, 429–431, 438–439, 693root, 172, 174, 303, 307, 421, 429,

430–431, 438, 464–465, 556,772–773, 775, 777, 779–783

starch granules, 202, 420–421, 428,432–433, 435, 703, 710–712, 715,719

stem, 168, 172, 180, 417–419, 421,424, 429, 556, 707, 710–711,713–714

storage structures, 435–436

940 Index

Botanical specimens (cont.)suspension-cultured cells, 189,

429–430tapetum, 433–434, 779waxes, 420, 428, 434–435, 714–715

point spread function in, 784refractive index heterogeneity, 192,

418–420maize stem, 419

Bovine embryo, 750.Boyde, Alan, 2, 6, 141, 154, 224. See also,

Stereoscopic images.BPNN. See Backpropagation neural

network.Bragg grating, tuning diode, 107.Brain slices, 392–398, 722–734. 686.

beam collimation, 728choice of objectives, 395, 727–728future directions, 929image processing for, 732–734

algorithms, 733alignment, center of mass in, 732–733alignment, based on image overlap,

732automatic detection of neurons,

733–734drift/vibration compensation, 396, 732image de-noising using wavelets, 734

image processing/analysis, 330–331,395–396, 730–732

biocytin protocol, 730classified using cluster analysis,

731–732correlated electron microscopy, 731montaging, 331neuron reconstruction, 330–331, 730protocol for PCA/CA, 731–732spectral imaging, 382two-photon/neurolucida system, 316

image production, 7292-photon excitation, 727deep imaging, 395living neurons, 725maintaining focus, 395, 732microglia, 397–398neuronal ensembles, 726objective lenses, choice of, 727–728second harmonic imaging, 729–730

in vivo observations, 387preparation, 387

labeling cells, 394–396, 724–727biolistic transfection, 724–725bolus injection, 726calistics, 726choice of dyes, 729diolistics, 726dye injection/patch clamp, 726genetic manipulation, 725–726GFP transgenic mice, 726Helios Gene Gun, 724live-dead staining, 393painting with AM-ester indicators,

726–737

photoactivation, 383slice loading, 726

linear unmixing, 192, 382, 664–667making brain slices, 393, 722–724

acute slices, 722–723autofluorescence, 383cultured slices, 724mouse visual cortex, 723primary visual cortex, 724protocols, 731thalamocortical slice, 724

photodamage, 729pulse broadening, 728reducing excitation light, 390–391resolution, 729second harmonic imaging (SHG),

729–730silicon-intensified target (SIT) camera

use, 730slice chamber, 394

protocol, 727speckle microscopy, 383useful techniques, table, 384–385time-lapse, 382two-photon imaging, 727

calcium imaging, 729z-sectioning, 729

Breakdown.electrical, in PMTs, 263, 660optical, high power density, 198, 680,

682, 685, 687, 703, 705Brewster surfaces, 83.Brewster windows, 83, 102–103, 115.Bright-field microscopy, 6, 127, 130, 201,

224, 229, 448, 468, 649, 728.CCD for, 127, 483deconvolution, 468, 472–473depth of field, 4low coherence light for, 130, 134–135,

139–140optical projection tomography, 610–612

Brightness, source, 21, 26, 126–127,129–130, 141–142, 215.

and exposure time, 141–142gray levels, 71–73as limitation of disk-scanners, 21, 215of non-laser light sources, 126–127of sun, 127, 135

Brillouin background, in glass fibers, 88.Brillouin effect, reduction, 110.Brownian motion, microtubules, 11.BSL. See Backscattered light.Buffering, fluorescent ion measurement,

740.Bulk labeling, in living embryos, 761.

CC. elegans, 746, 748, 766, 856, 857–858.

cryopreparation, 857–858FRET imaging, 766as model system, 746, 748TEM images, 856, 857

Ca2+ imaging, see Calcium imaging.

Ca2+ indicators, 346–347, 738, 742–743. See also, Ca2+ sparks, 737–738, 742.

discovery, 737, 738Caenorhabditis elegans. see C. elegans.Caged compounds, 759–760.

multi-photon excitation, 543–544Calcein AM dye, 355, 360, 362–363,

426–427, 430, 685, 804, 812.Calcium imaging, 529, 545, 584, 736–737,

812.calibration, 742–743data compression, 584intensity image, 529introduction, 736multi-photon excitation, 545ratiometric, 189signal-to-noise ratio, 737single-cell kinetic, 812TIRF for measuring, 180very fast imaging, 237

Calcium ion dyes, 183, 189, 237, 736, 737,741–743. See also, fura-2, Fluo-3and Indo-1.

Fluo-3 and Fura Red indicator system fordetermining, 183

Fluo-3 indicator system for determining,737

fura-2 reactions, 741–742Indo-1 and Fura-2 indicator system for

Calcofluor, 424, 438.staining procedure, 438

Calibration, 34, 75–76, 742–745.Ca2+ sparks, 742of CCD to measure ISF, 75–76confocal microscopy, 742errors in, 744of ion concentrations, 742–745ion interference, 745of effective pinhole size, 34in vitro, 742

Calistics, 726.Callus, 429.Cambridge Technology, galvanometers,

54.cAMP indicators, 347.Canna, 422, 710.

fluorescence spectra, 422as function of excitation intensity, 165nonlinear absorption, 710

Carbon arc lamps, 136.CARS. See Coherent anti-stokes Raman

scattering.CARS correlation spectroscopy (CS-CARS),

602.Raman spectra, 602

CARV disk-scanning confocal microscope,215, 226, 229, 230, 907–908.

diagram, 230, 907CAT. See Computed axial tomography.Cathode-ray tube (CRT), 5–6, 53, 67,

72–73, 291, 293, 588–589.gamma, compensation, 73

Cavities, of dielectric coatings, 46, 47.

Index 941

Cavity-dumped lasers, 111, 114.for FLIM imaging, 114

CCD. See Charge-coupled devices.CD. See Compact disks.cDNA-GFP fusion, in plants, 773.Cedara, 281–282, 288, 302, 308.Cell adhesion imaging with TIRF, 90.Cell autofluorescence, 742.Cell chambers, 11, 22, 191, 219, 370–371,

386–387, 394, 429–430, 564,610–611.

for 4Pi confocal, 564for biofilms, 870–873, 875, 877, 880, 885brain slice, 394, 723, 727, 729for epithelial cells, 370–371, 377, 386finder chamber, 683flow chamber, 870–873, 875, 877, 880,

885for high-content screening, 810for optical projection tomography,

610–611perfusion, 394for plant cells, 191, 429–430simple, 22, 394for SPIM, 613, 625, 673table of required functions, 380table of suppliers, 388–389test chamber/dye, 654, 661

Cell cycle, 790, 791.Cell damage, 2-photonmicroscopy, 680–688

See also, Bleaching; Photodamage.absorption spectra of cellular absorbers,

681intracellular chromosome dissection, 688mitochondria, 686nanosurgery, 219, 686–687one-photon vs. multi-photon, 680–689by optical breakdown, 198, 680, 682, 685,

687, 703, 705photochemical, 682–685

absorbers/targets, 682beam power sensor, 683impact on reproduction, 686, 685laser exposure parameters, 682–683NIR-induced DNA strand breaks,

683–684NIR-induced ROS formation, 683photodynamic-induced, 684spectral characteristics, table, 682

photothermal, 685reproductive effect, short NIR pulses,

682, 686ultrastructure modifications, 685–686

Cell microarray (CMA), 815–816.Cell motility, 757.Cell nuclei, optical effects, 23.Cell pellet, three dimensional, 815.Cell surface targeting assays, 813.Cell walls of plants, 168–169, 188–189,

303, 306, 416–417, 420–421,428–431, 435–436, 438, 439,710–711, 713–715, 719, 769–776,779–782.

labeling, 775viability, 780

Cell-by-cell analysis, 817.Cell-cell signaling, 778.Cellular structures, optical effects, 22–23.Center-of-mass alignment protocol, 733.Center pivot/off-axis pivot mirrors, 1, 214.Cerium, doping of quartz lamp envelope,

116.CFP and YFP molecules, in FRET pair,

798–800.Chambers for living cell imaging, 388–389.

commercial suppliers, table, 388–389Charge amplifiers, 923–924.

defined, 923destructive readout, 923FET amplifier performance, 923non-destructive (skipper), 923

Charge-coupled device (CCD), 26–28,30–31, 39, 61–62, 65, 70, 74–78, 88,127, 137, 142, 215, 233, 254,458–459, 460–461, 482, 552, 558,644, 754–755, 784, 918–931. Seealso, Electron-multiplier

CCD.bit depth, 75camera, 918–931

advances in, for speed, 754–755bright-field imaging, 127for disk scanner systems, 78, 205, 215,

220, 233–235, 349, 459, 754–755pixel size, 62, 65, 634–635, 784specifications, table, 929time for sampling, 70

choosing,color, 927computer-assisted pulse shaper, 88confocal imaging, 458–459cooled, advantages and limitations, 30–31

quantum efficiency, 26–28spatial quantization of signal, 39

digital camera, 75digital vs. video camera, 61–62electron multiplier-CCD, 30–31, 76–77,

233–235, 262, 459–461, 482,925–926

multiplicative noise, 77, 234, 257, 262,926

result, 205, 234, 755table, 233, 459

evaluating, 927–931array size, 928–929“the clincher,” 929comparison, CCD/EM-CCD, table,

233, 459dynamic range vs. pixel size, table, 928maximum signal, 930quantum efficiency, 927–928readout noise, 928readout speed, 928–929self test, 930sensitivity, 930shutter stability, 929

specifications, 927, table, 233, 929user-friendliness, 929

gain-register, 76–78, 460–461intensified, 930–931. See also, Intensified

CCDmonitoring during exposure, 137multi-focal multi-photon microscopy, 552,

558noise sources, 256, 924–925

charge amplifier, 925clock-induced charge (CIC), 234, 926fixed pattern noise, 924–925multiplicative noise, 77, 234, 257, 262noise vs. pixel dwell time, 922table, 256

operation, 254, 918–927blooming, 923charge amplifiers, 923–924charge coupling, 918–920charge loss, 921dark charge, 921–922destructive readout amplifiers, 924edge effects, 921electron multiplier, 926–927FET amplifier performance, 253, 922,

924frame transfer readout, 920full-frame readout, 920gain register amplifier, 925–926incomplete charge transfer, 923interline transfer readout, 920leakage, 921–922non-destructive (skipper) amplifiers,

923–924possible problems, 920quantum efficiency vs. wavelength,

922quantum efficiency, 920–921readout methods, 920signal level representing zero photons,

925storage array, 920

performance, table, 256, 459, 923piezoelectric dithering, increases

resolution, 70pixel size, 62, 65, 634–635, 784, 928quantum efficiency and noise, 29, 644,

920, 922measuring, 74–76, 926

sensors size, parallel data collection, 142snapshot camera, 65specifications, described, 927–930testing, 930

Cheek cells, backscattered light image,22–23.

Chemical environment probe, 517.Chimeric fusion proteins, 794, 801–802.

anisotropy analysis, 794cloning for FRET, 801–802overexpression, 802

Chinese hamster ovary cell, 197, 556. 684+,818.

Chirp, pre-compensation, 88, 111, 602, 907.

942 Index

Chlorophylls, autofluorescence, A. thaliana,175, 194, 203, 425–426, 528, 711,714, 779, 782, 881.

bleaching, 203FLIM, 528

Cholera toxin transport, 790–791, 796–797,802.

FRET, 796–797, 802–803Chromatic aberrations, 134, 152–156, 178,

242–245, 657–658, 659.apparatus for measuring, 243axial chromatic registration, 243–345,

658, 657–659of incandescent and arc lamps, 134intentional, for color/height encoding, 154lateral chromatic registration, 657–658fluorescent latex bead labeled, 178linear longitudinal chromatic dispersion,

154, 659, 664measuring, 242–245

Chromatic corrections, 157, 177.excitation/emission wavelength, 177tube length, table, 157

Chromatic magnification difference. SeeLateral chromatic aberration.

Chromatin, 385, 390, 684, 693–695, 812.Chromophores, 338–348, 543–544,

803–804. See also, Dyes;Fluorophors; Fluorescent probes etc.

cellular introduction methodselectroporation, 359–360, 803microinjection, 360–361, 388, 739,

748, 755, 795, 803–804table, 344–345, 803transfection reagents, 358, 360, 362,

556, 682, 790–791, 795, 803multi-photon excitation, 543–544

CIC, clock-induced charge, EM-CCDs, 234,926.

Circular exit pinhole, 9.Circular laser beam, corrective optics, 106.Classification, pattern. See Automated

interpretation of subcellular patterns.Clathrin-GFP dynamics, 236.Clearing agents. See also, Mounting media.

optical projection tomography, (OPT)610, 624

plant material, 166, 417–420, 439,774–775

Clock, role in digitizing and reconstructinganalog signal, 64.

Clock-induced charge, in EM-CCDs, 234,926.

Closterium, 192–194.chloroplast autofluorescence, 192–195signal variation with depth, 194

CLSM. See Confocal laser-scanningmicroscopy.

Cluster analysis (CA), 731–732, 826.neurons classified using, 731–732protocol with PCA and, 731–732subcellular patterns, 826

CMA. See Cell microarray.

CNS, (central nervous system), 392–393,395. See also, Chapters 19 and 41.

Codecs, image processing, 831, 836,840–841.

Coefficient of variation, 660, 661.Cohen’s k statistic, 826.Coherence length, 7–8, 84.

defined, 7–8, 84, 130–131reducing, for laser light, 84

Coherence surface, 84.Coherence volume, 84.Coherent anti-stokes Raman scattering

(CARS), 90, 204, 550, 595–605.advantages, 204, 596correlation spectroscopy, 602–603defined, 595energy diagram, 596epi-detected, 597–599forward/backward detected, 597–599Hertzian dipole radiation pattern, 598history, 595–596imaging of biological samples, 603–604

adipocyte cells, 604artificial myelin, 204epithelial cells, 603erythrocyte ghosts, 603

intensity distribution, 597mapping intracellular water, 90microscope schematic, 599multiplex CARS microspectroscopy,

601–602non-resonant background suppression,

600–601energy diagram for multiplex CARS,

601epi-detection, 600phase control of excitation pulses, 600picosecond vs. femtosecond pulses,

600polarization-sensitive detection, 600time-resolved CARS detection, 600

optimal laser sources, 599–600pumped optical parametric oscillator

(OPO)systems, 600

perspectives on, 604–605unique features under tight-focusing,

596–597Coherent illumination, 1, 83–84.

properties of laser light, 83–84and resolution, 1

Collagen fibers, 164, 188, 313, 361, 393,514, 703–704, 715.

autofluorescence, 545birefringence, 164, 188, 717gels, 393polarization microscopy, 164, 188second harmonic image (SHG), 703–704,

715Collector optics, elliptical and parabolic,

129.Colliding-pulse, mode-locked laser (CPM ),

540.

Colloidal gold labels, 167, 241, 846–859.contrast, 167electron microscope markers, 846–857

correlative, 850, 852, 855SEM, 850TEM

FluoroNanoGold, 854GFP related, 854–855, 857–858measuring resolution, 241quenches fluorescence, 854Rayleigh scattering, 167

Colocalization, 517, 650, 667–670, 794,813, 881.

FRET, FRET, 519erroneous, 581

Color display, 291, 292.display space, 291multiple channel display, 292palette, 291pseudo, 173–175, 190, 291resolution, 291true, 291

Color centers, in optics, avoidance, 116.Color filters, 43–52. See also, Filters.

long-pass, 43–46, 175, 203–204, 212short-pass, 45, 46bandpass, 44, 45

Color print images, 592.Color reassignment, 173–175, 190, 291.Coma, 145, 151, 245, 247, 249, 483,

630.distortion away from optical axis, 151observation using point objects, 145,

246Commelina communis, images, 712.Commercial confocal light microscopes,

906–917.BD-CARV II, 230, 907La Vision-BioTec TriM-Scope, 907Leica, TCS SP2 AOBS, 910Leica MP RS Multi-photon, 910Nikon C1si, 911Olympus DSU, 913Olympus Fluoview-1000, 912optical parameters of current, table,

908–909Visitech VT Infinity, 914Visitech VT-eye, 914Yokogawa CSU 22, 231, 915Zeiss LSM 510 META optical, 916–917Zeiss LSM-5-LIVE Fast Slit Scannerschematic, 232, 916

Compact disks (CD) for data storage, 499,586–587, 588, 731.

Compact flash cards, 588.Components, of confocal fluorescence

microscopes, 43–58, 207–208.acousto-optical devices, 54–57chapter, 43–58electroptical modulators, (Pockels cells),

25, 54, 57, 87, 116, 543, 701,903–904

filters/beam-splitters, 44–51

Index 943

mechanical scanners (galvanometers),51–54

polarizing elements, 58Computed axial tomography (CAT),

610–611.Compression, data see, Data compression.Condenser lens, size, 129.

magnification, 128–129Configuration of pixels in image plane, 62.ConfMat. See Confusion matrix based

method.Confocal disk-scanning microscope. See

also, Disk-scanning confocalmicroscopy.

Confocal fluorescence microscope, 73, 207,404–413. See also, Confocalmicroscopy; Confocal laser-scanningmicroscopy.

basic optical layout, 207limitations due few photons, 73, 459refractive index mismatch, 404–413 See

also, Refractive indexConfocal imaging, 4–5, 232, 235–236, 737,

738, 746–766, 809–817. See also,next major head and

Chapters 35 and 36.4Pi. See 4Pi microscopyautomated

for cytomics chapter, 809–817of microarray slide, 816platforms used for, 810real-time, 810temperature control, 810types of assays for, 811, 813–814workstations, 814

of biofilms, Chapter 50deconvolution, 753. See Deconvolution

by disk-scanning confocals, 232fast, 235–236of fluo-3 loaded cardiac myocyte, 737fluorescent indicators for, 738high-resolution datasets, cell

arrangements, 776of living cells, 813of living embryos, chapter, 746–766methods compared, 459, 644–647. See

Chapters, 22, 23, and 24of plants, 773. See also, Chapters 21and

43vs. non-confocal, 746time-lapse. See Time-lapse imaging

Confocal laser-scanning microscopy(CLSM), 9–15, 32, 38, 81, 89, 118,222–224, 408, 518, 678, 690, 697,750–751, 754, 884–885. See also,next major head

advantages and limitations, 11–12,222–223, 644–647, 884–885

alternatives to, 644–647, 754comparisons, 644–647disk-scanning and scanned slit, table, 224digitizer employing full integration for, 32edge response, 408

fluorescence lifetime imaging, chapter,518

laser power required, 81laser requirements for, 89vs. multi-photon laser-scanning

microscopy, 750–751. See Chapters22, 23, 24

photobleaching, 690, 697vs. selective plane illumination

microscopy, (SPIM), 678stage-or object-scanning, 13–15TEM mode, 118zoom magnification and number of pixels,

38Confocal microscopy, 90, 141, 265,

381–399, 444–447, 453–467,650–670, 742, 770, 774, 779, 810,811, 815, 870–887. See also,preceding major head and Chapters35 and 36.

art of imaging by, 650automated, platforms used for, 810balancing multiple parameters for, 650of biofilms, 870–887calibration of, 742cell microarray and, 815colocalization, 667–670

effect of MLE and threshold, table, 669fluorogram analysis, 669image collection, 667–668nerve fiber, 669quantifying, 668setting thresholds, 668spatial deconvolution in studies,

668–670vs. deconvolution, 644–647, 453–467. See

also, Chapters 22, 23, 24CCD/confocal imaging combination,

458–459deconvolving confocal data, 461–464,

466, 488–500fluorescence excitation, 459fluorescent light detection, 459–460gain register CCDs, 460–461image sections, figures, 455, 456, 462imaging as convolution, 453–457integration of fluorescence intensity,

459limits to linearity, 457model specimens, 461noise, 459–463out-of-focus light, 461point spread function, 453–457practical differences, 458, 463–466resolution, 459–463same specimen comparison, 465sensitivity, 459–463shift invariance, 457, 490, 564single point imaged, 454summary of pros/cons, table, 459temporal resolution, 458

focus positioning, 651–652getting a good confocal image, 629–631

alignment of optics, 629–630back-focal plane (BFP), 210, 509, 629,

633focus, 629low signal, 631mirror test specimen, 630no signal, 631, 660simultaneous BSL/fluorescence, 631

high-content screening systems, table, 811illumination sources, 126–144, 650–651

See also, Lasers; Non-laser sourcesacousto-optic tuning filter (AOTF),

651.laser sources, chapter, 80–125laser stability, 651power measurement, 650–651

living cells, 381–399. See also, Livingcells

Minsky first confocal design, 2, 4–6, 11,141, 216, 890

monitoring instrument performance,650–663

illumination source, 650–651optical performance, 652–660photon efficiency, 14–15, 24scan raster/focus positioning, 651–652signal detection, 660–663

with non-laser light, 141objective lens, 652–660. See Chapter 7optical performance, 652–660. See also

Chapters 7, 11axial chromatic registration, 658–659axial resolution vs. pinhole, 656–657.

See also, Axial resolutioncontrast transfer function, 656. See

CTFcoverslip thickness and RI, table, 654field illumination, 658flatness of field, 659Focal Check™ beads, 657–659lateral chromatic registration, 657–658lateral resolution, 655refractive index, 654. See Chapter 20resolution test slides, 656self-lensing artifacts, 659spherical aberration, correction, 654,

655subresolution beads, 655–656x-y and z resolution using beads, 656

optimizing multi-labeling, 663–667bleed-through between channels, 663control samples, establishing limits,

663measuring autofluorescence, 663multi-tracking, reduces bleed-through,

664positively labeled sample, 664reflected light contribution, 663secondary conjugate contribution, 664

photon efficiency, 24, 26, 28, 30 33–34,36

polarizing elements, 57scan raster, 651–652

944 Index

Confocal microscopy (cont.)malfunctioning system, 653phototoxicity from uneven scan speed,

651sources of fluorescent beads, table, 653well-calibrated system, 652x and y galvanometers, 651–652z-drive mechanism, 652z-positioning calibration, 654z-positioning stability, 652

separating signal by spectral regions for,664

sequential collection reduces bleed-trough, 664

signal detection for, 660–663coefficient of variation, 660–661instrument dark noise, 660PMT linearity, 661–662signal-to-noise ratio, 660spectral accuracy, 662spectral detector systems, 662spectral resolution, 662–663wavelength response, 663

signal level, 444–445signal-to-noise ratio, 444–447spectral analysis, of plants, 770spectral unmixing, 192, 382, 664–667

limitations to, 667overlapping fluorophores separation,

664–667removing autofluorescence, 667

stage-scanning, 9staining plant cells, 774vs. structured-illumination methods, 265vs. two-photon excitation, 779

Confusion matrix based method (ConfMat),826.

Constant output power laser stabilization,86.

Continuous wave (CW) laser, 87, 88,90–118.

beam intensity stabilization, 86–87diode (semiconductor), 105–110

output power/cooling, 108pumped solid-state, table, 94, 95

dye lasers, 86, 103, 112, 114, 124,540–541

fiber up-conversion, 109–110gas lasers

Argon-ion, 90, 102Krypton-ion, 102HeNe, 102HeCd, 103cesium and rubidium vapor, 103–105

table, 92–93titanium-sapphire, 109

Contrast, 7, 11, 16, 37, 39, 49, 59–62, 68,159, 162–204, 248, 421, 473, 488,542, 599–600, 607, 622, 656, 657,675. See also, Rose criterion andCTF.

absorption, equations, 164chapter, 162–206

defined, 162flare, 649formation of, chapter, 162–206fluorescence. See Dyes, and Fluorophores

as function of feature size, 16,61–62, 37, 634

intrinsic, 633measuring, 16, 59polarization. See Polarization microscopysecond harmonic generation. See SHG

and statistics, 633third harmonic generation. See THG

Contrast medium, and laser power, 80–81.Contrast method, defines signal required,

126.Contrast transfer function (CTF), 16, 35,

37–39, 59–62, 656, 747.in confocal vs. non-confocal microscopy,

16.See Chapter 11as function of grating period, 16of microscope optical system, 35relationship with objective BFP, 61and spatial frequencies, 16, 37and stages of imaging, 62

Control, of non-laser light sources, 138–139.Convalaria majalis, 425, 556.

fluorescence microscopy of rhizome, 425multi-focal multi-photon imaging, 556

Conversion techniques, 259–260.analog-to-digital, 259digital-to-analog, 259–260

Convolution, a primer, 485–487.3D blurring function, 486Fourier transforms, 487geometrical optics, 487out-of-focus light, 486–487

Cooling water, checking/maintaining,116–117.

Cork microstructure, 770.Correction collar, (spherical aberration), 15,

145–149, 158, 160–161, 178,241–242, 247, 377, 407, 410–412,471, 654–655, 657.

adjustment, 377, 407, 471, 499, 654–655dry objectives, 410multimedia, 640

Correctors, 70, 147.spherical aberration, 15, 151, 147, 192,

411–412Intelligent imaging innovations, 78–79,

151, 192, 395, 411, 654to stored data, second Nyquist constraint,

70Corrective optics, for diode lasers, 107–108.Correlational light microscopy/electron

microscopy, 731, 434, 436–437,846–860.

BSL image, 855brain slices, 731then CLSM, 856–857cryopreparation of C. elegans, 857–858DIC image tracking, 849DIC image/UV fluorescence image, 850

different requirement of LM/EM,846–850

early 4D microscopy, 846fluorescence/TEM to analyze

cytoskeleton, 854fluorescent micrographs, 851FluoroNanoGold for cryosections, 854GFP, 854. See also, Green fluorescent

proteinHVEM stereo-pair, 848–849immuno-stained bovine aorta, 852LVSEM of FRAPed microtubules, 849,

850phalloidin as correlative marker, 235–236,

344, 376, 378, 694, 696, 756, 804,854–856

phase-contrast imaging, 851postembedding, 855quantum dot labeling, 853same cell structure LM/SEM, 850–852same cell structure LM/TEM, 852–856SEM images at 5kV and 20kV, 847, 848TEM cross-section of C. elegans, 856TEM longitudinal section of C. elegans,

857tetracysteine tag labeling, 221, 348, 357,

853tiled montage TEM images, 858time-series DIC images, 847

Correlative LM/EM. See Correlational lightmicroscopy/electron microscopy.

Coumarin dye, 114, 339, 344–345, 353, 355,654–655, 661, 693.

Counting statistics, 20, 30. See Poissonstatistics.

Cover glass. See Coverslip.Coverslip, and spherical aberration, 15,

147–150, 201. See also, Sphericalaberration.

CPM laser. See Colliding pulse mode-lockedlaser.

Crane fly spermatocyte, metaphase spindle,15.

Creep, in piezoelectric scanners, 57.Cr:Fosterite, femtosecond pulsed laser, 109,

114, 415, 541, 706–709, 712–714.Critical angle, for reflection of incident light

surface of refracting medium, 167,502.

Critical illumination of the specimen,128–129.

Crosslinking fixatives, 369.Crosstalk.

between fluorescence channels, 203, 424,882

between disk pinholes, 227between excitation foci, 553–556,

558–559, 564CRT. See Cathode-ray tube.Crystal Fiber A/S, HC-800-01 bandgap

fiber, 88.CSU. See Confocal scanning unit.CTF. See Contrast transfer function.

Index 945

Curtains, laser, safety, 118, 904.Cuticles, plant, 434–437, 715, 717, 779.

insect, 166maize, 436

CW. See Lasers, continuous wave.Cyan fluorescent protein (Cyan), 221–222.Cyanine dyes, 339, 342, 344, 353–355,

362–363, 374, 443, 540, 587, 760,854, 874.

Cytomics, 810, 811.automated confocal imaging, 810automated confocal imaging, table, 811

Cytoskeletal structures, 24, 188, 190,328–329, 368, 370, 372, 378, 383,461–462, 577, 703, 715, 719,773–774, 813, 846–848, 852, 854.

LM-TEM analysis, 846, 854stabilizing buffer, 852

Cytosolic markers, 757.Cytotoxicity, reducing, 36–37. See also,

Bleaching; Phototoxicity.

DDAC. See Digital-to-analog converter.Damage threshold, LED sources, 139.DAPI, 140, 344–345, 355, 358, 376, 431.

plants, 431use of, 376

Dark current, 29, 76, 234.fixed-pattern noise due to, 76of photomultiplier tube, 29, 660reducing, 234

Dark noise, defined, 232.Darkfield microscopy, 5, 7, 172, 474, 672.

depth of field, 4Data, 11–12, 33, 64, 76, 237.

conversion from ADU to electron data, 76degradation by multiplicative noise anddigitization, 33reconstructing, 64speed of acquisition, 11–12storage of volume of data, 237

Data collection guidelines, 319–320.Data collection model, blind deconvolution,

472.Data compression, 288–289, 292–293, 295,

319, 499, 580–585, 762, 764, 819,835–836.

algorithms, 580discrete cosine transform (DCT), 581Huffman encoding, 580Lempel-Ziv-Welch (LZW), 580run-length encoding (RLE), 580

archiving systems, 580gzip, 580PKzip, 580WinZip, 580

calcium imaging, 584color images, 581different techniques, table, 581Dinophysis image, 585effects on confocal image, 583examples, 583–585, 592, 834–837

file formats for, 580–588fractal compression, 581–582GIF (graphics interchange format),

580JPEG (Joint Photographic Experts

Group), 581–584MPEG, 836–839, 840–841PNG (portable network graphic), 581,

584QuickTime, 829, 831, 836–837,

840–844TIFF (tagged image file format), 580wavelet compression, 581–584, 819

movies, 836–842artifacts, 839compression ratios, 842–843entrope, 841MPEG formats, 840–841Up-sampling, 838

pixel intensity histograms, 584testing, 830, 835time required, table, 581for WWW use,

816useful websites, 844–845

Data projectors, 590.Data storage, 106. See also, Mass storage.Data storage systems, 287, 395. 580, 594,

764.chapter, 580–594characteristics of 3D microscopical data,

287databases, 861–869. See Databasesrandom access

CDR, CDRW, 586–587DVD, 587Magnetic disks, 586semiconductor, FLASH memory, 588

for remote presentation, 842role for STED, 577

Databases, 2D/3D biology images, 827,861–869.

benefits, 863–864fast, simple machine configuration, 863improved analysis and access, 863performance, 863remote monitoring, 863repeatability of experiments, 863submissions to other databases, 863

criteria/requirements, 866–867digital rights management, 867metadata structure, 867query by content, 866–867user interface, 866

data/metadata management, 861–862future prospects, 867image database model, 864–865image information management, 862image management software, table, 865,

868instrument database model, 864laboratory information management

systems (LIMS), 862microscopy data/metadata life cycle, 863

modern microscopes design aims,862–865

projects, 865–866BioImage, 865–866Biomedical Image Library (BIL), 866Scientific Image DataBase (SIDB), 866

recent developments, 861–862MPEG-7 format, 862relational database management

systems (RDBMS), 862TIFF format, 861

software for, 868–869ACDSee, 868Aequitas, 868, 869Cumulus, 868Imatch, 868, 869iView, 868, 869Portfolio, 868price, 868Research Assistant, 868ThumbsPlus, 868, 869

system requirements, 864DBR. See Distributed Bragg reflector.DCT. See Discrete cosine transform.Deblurring algorithm, 476.Deconvolution, 7, 26–28, 39, 40, 66,

189–190, 222–223, 278, 456–458,464, 468, 488–500, 542, 564, 736,746, 751–753, 778, 784–785, 828,864, 900, 929. See also, Blinddeconvolution.

of 2-photon images, 498and 3D Gaussian filtering, 70, 281, 285,

323, 392, 395, 667. See also,Gaussian

4Pi lobe removal, 562, 565advantages and limitations, 458, 475algorithms, 472–476, 490, 495–497, 751,

778comparison, 497–498iterative constrained Tikhonov-Miller,

497Jansson-van Cittert, 496nearest neighbor, 495–496non-linear constrained iterative,

496–497Richardson-Lucy, 497, 568Weiner filtering, 496

background history, 488–490blurring process contributions, 488equation showing restoration possible,

489image formation, 489–490schematic diagram of convolution, 489

blind, 189–190, 431, 463, 469, 472–473,478, 486, 492, 496–497, 646

chapter, 468–487maximum likelihood estimation,

472–477, 483, 669–670blurring process contributions, 488confocal data, 39, 40, 453–467, 488–500,

753, 778. See also, Confocalmicroscopy, vs. deconvolution.

946 Index

Deconvolution (cont.)of simulated confocal data, 40

CARS data cannot be deconvolved, 397,399

chapter, 453–467colocalization, 668–670 comparison of

methods, 66, 453, 467, 475–477,497–499, 644–648

convolution primer, 485–478convolution and imaging, 490–491

Fourier transform of PSF, 489, 490linearity, 490optical transfer function, 490–491shift invariance, 457, 490, 564

and data compression, 584–585test results, 401, 461, 464–466,

481–482, 483defined, 189–190, 468display of data, 301, 830, 835–836examples, 40, 190, 392, 411, 462, 466,

471, 488–498, 5104Pi, 468, 565botanical specimens, 784–785brightfield, 411, 475, 478cardiac t-system, 498confocal, 470DIC, 470polarized, 479of simulated confocal data, 40STED, 574–576

flatfielding the data, 477black reference, 76white-reference, 76

fluorescence lifetime imaging, 521four dimensional deconvolution, 391–392,

752Fourier transform of PSF, 489, 490future directions, 483, 766and image formation, 490–492linearity and shift-invariance, 457, 564live imaging, 480, 564, 751–754missing cone problem, 494model specimens, 461, 464–466,

481–482, 483multi-photon, 488–500, 542multi-view montaging, 330, 677

ion imaging, 736noise, 495, 635and Nyquist reconstruction, 59, 65, 67,

68, 222–223, 635suppressing Poisson noise, 39

optical sectioning, 752out-of-focus light, 26–28, 431, 487, 644and pinhole, 26, 487point-spread function (PSF), 223, 241,

247, 453, 463, 471, 489–492, 635,655

approximations, 493measuring PSF, 492–494

and Poisson noise reduction, 320pre-filtering, 281, 497, 581problem with specimen heterogeneity, 22,

648

purpose, 468requirements and limitations, 489–494

diagram demonstrating convolution,489

linearity, 490missing cone problem, 494noise, 495optical transfer function, 490–491point spread function, 489, 492–494shift invariance, 457, 490, 564

sampling frequency, 635spherical aberration, 471, 480–481stain sparsity, 28structured illumination, comparison,

265–279subpixel refinement, 478–479temporal/spatial, 392, 458, 753transmitted light imaging, 472, 475,

478of wavelength spectra, 382, 663–667,

771–772limitations, 667

Deconvolution lite, 68–70.Deflector, acousto-optical. See Acousto-

optical deflector.Defocusing, size and intensity distribution,

146.Degree of modulation, 268–270.

locally calculated, 268–270absolute magnitude computation,

268–269equations, 269homodyne detection scheme, 268–269max/min measured intensity difference,

268scaled subtraction approach, 269–270square-law detection, 268–269synthetic pinholes, 268

Delamination, and interference fringes,168–170.

Delivery, dye, 355, 357–360, 810.Deltavision, 132, 282.Demagnification, and numerical aperture,

127.Depth discrimination, in LSCM. See Axial

resolution.Depth of field, 4, 9, 13.

extended-focus images, 9fluorescence microscopy, 4phase-dependent imaging, 13

Depth-weighting, projection images, 304,306.

exponential, 304linear or recursive, 304

Derived contrast (synthetic contrast),188–201.

Descanned detection, 166, 208, 212, 537,540–542, 754, 904.

Design of confocal microscopes, 43, 145,166, 207–211, 237. See also,Commercial confocal lightmicroscopes.

4Pi, 563, 566

of confocal fluorescence microscope, 208efficiency in, 43fast-scanning confocal instruments, 237intermediate optical system, 207–209of microscope optics, 145MMM, 552, 555practical requirements, 210–211of transmitted confocal microscope, 166

Detection efficiency, 34, 35, 210–211.measurement, 34–35practical requirements, 210–211

Detection method, multi-photon, 541, 542.descanned, 542

Detectors, 9, 11, 25, 28, 251–264. See also,Photomultiplier tube; Charge-coupled device, etc.

area detectors. See Image detectorsassessment of devices, 260–262CCD, 254

noise vs. pixel dwell time, 922comparison, table, 255–256conversion techniques, 259–260descanned, 208, 212, 537, 540–542, 774,

904direct effects, 252errors, 211–212evaluation, 211–217future developments, 262–264history, 262–264image dissector, 254–255image intensifier, 13, 232–233, 519–520,

524, 555–556gated, 233, 519–522, 524, 555–556intensified. See Intensified CCD

MCP-PMT. See Microchannel platemicrochannel plate, 232–233, 255, 262

MCP-CCD, 262gated, 519, 523–524, 527, 532

noise internal, 256–259internal detection, 256noise currents, table, 256photoemissive devices, 256–257photon flux, 257–258pixel value represented, 258–259

non-descanned, 185, 201, 218, 381, 447,456, 507, 542, 552, 559, 643, 646,727, 750, 779, 904, 909–910

phase-sensitive, 518–520, 619photoconductivity effects, 252, 253photoemissive, 254photography. See Photographic systemsphotomultiplier tube, 9, 11. See also,

PMTphotovoltaic effect, 252–253photon interactions in, 252–256point detectors, 260–261quantal nature of light, 251–252quantum efficiency (QE) vs. wavelength,

25for second harmonic detection, table, 707silicon-intensified target (SIT) vidicon,

730spectral, 203–204, 662–663, 666–667

Index 947

TCPSC, 518, 520–523, 526time-gated, 522thermal effects, 252work functions, table, 252–253vacuum avalanche photodiode, 254, 255

Developmental biology, 545, 624.multi-photon microscopy (MPM), 545

Dextran labeling, 173–174, 292, 512, 757.DFB. See Distributed feedback.4¢,6-diamidino-2-phenylindole, 140,

344–345, 355, 358, 376, 431. SeeDAPI.

plants, 431use of, 376

Diatom, 438, 638–640, 881.as standard for measuring objectives, 145test specimen, 638–640

DIC. See Differential interference contrast.Dichroic filters, 212.

intensity loss, 212transmission, 212

Dichroic mirrors (beam-splitters), 44, 45,50–51, 129, 211, 217–218.

coating for collection mirrors, 129double and triple, 217–218effect of deflection angle, 211separating emission/excitation, 44–45,

50–51Die, of light-emitting diode, 133, 134.Dielectric butterfly, galvo feedback, 54.Differential interference contrast (DIC)

imaging, 10, 14, 76, 127, 146, 171,453, 468, 473–475, 846.

blind deconvolution, 473–475converting phase shifts to amplitude, 171narrow bandpass filter use, 76Nomarski DIC contrast, 2, 268, 746, 892photon flux reduction, 127schematic for, 475three dimensional, 470Wollaston prism, 156, 468, 473, 475

Diffraction, 61, 65.contrast transfer function, 16, 35, 37–39,

59–62, 656, 747and sharpness of recorded data, 65

Diffraction limit, 210–211. See also,Rayleigh criterion.

defined, 210point-spread function, 146practical requirements for, 210–211

Digital light processor (DLP), projectors,590.

Digital memory system, 64.Digital microscopy, optics/statistics/

digitizing, 79.Nyquist sampling, 146

Digital printers, 591–593.Digital processing, in disk-scanning

confocal, 12.Digital projectors, 590.Digital rights management (DRM), 830,

844.Digital video disks (DVD), 587–588.

Digital-to-analog converter (DAC), 64,259–260.

operation, 64Digitization, 25, 31–32, 36, 38–39, 59,

62–63, 66, 72, 75, 79, 259, 261, 286,460, 495, 639, 911.

aliasing. See Aliasingblind spots, 38and Nyquist criterion, 38–39precision, 25and pixels, 62–63of voltage output of photomultiplier rube,

31–32DiI derivatives, 760.Dimethylsulfoxide (DMSO), 697, 726–727,

760, 875.handling, 739

DIN standard, microscopes, 156.Dinophysis image, 585.Diode injection lasers, 105–108.Diode lasers, 86, 87, 107, 112, 116.

distributed feedback, 107emission stability, 86intensity, 87maintenance, 116modulated, 112noise sources, 86physical dimensions, 106violet and deep blue, 107visible and red, 107wavelength stabilization, 87

Diode-pumped alkali lasers (DPAL),103–105.

Diode-pumped lamp (DPL), 108–109.Diode-pumped solid-state lasers (DPSS),

108–109, 111, 112.kits, companies offering, 109passively mode-locked, 111ultrafast, 112

Diolistics, ballistic gene transfer, 726.Dipping objective, 149. 161, 209, 411, 429,

568, 613, 727, 737, 870, 872.Direct permeability, 358–359.Discrete cosine transform (DCT), 581.Disk-scanning confocal microscopy,

215–216, 224, 225, 228–229,234–235 754, 755.

advantages and limitations, 223–224for backscattered light imaging, 228–229chapter, 221–238commercial instruments, 907, 913, 915comparing single- vs. multi-beam, 224

table, 226and electron multiplier CCDs, 78, 205,

215, 220, 233–235, 349, 459,754–755

embryo, 754high-speed image acquisition, 216,

222–224, 754image contrast in, 168–171microscopes, table, 224optical sectioning, 235types, 228–232

Dispersion, optical, 56, 88, 152, 154, 242,411, 542–543, 609, 683.

in acousto-optical devices, 3, 15, 55–56,88

CARS signal generation, 728compensation, 566–567defined, 152in fiber lasers, ultra-fast pulses, 88, 110,

113by filter blank material, 211generates third harmonic signal, 704–705group delay dispersion, 537–538, 543group velocity dispersion, 88, 111, 210,

537, 609, 903in optical coherence tomography, (OCT),

609in optical fibers, 502, 504, 507and temperature, 15, 411for multi-channel detection, 51using to correct for chromatic aberration,

153Display software. See Presentation software.Displays, 580, 588–590, 594, 892.

cathode ray tube (CRT), 5–6, 53, 67,72–73, 291, 293, 588–589

data projectors, 590digital light processor (DLP), 590

halftoning vs. dithering, 589international television standards, 589liquid crystal (LCD), 589–590

supertwisted nematic (STN), 589thin-film transistor (TFT), 589

monitors, 588–589Distortion, 39–41, 152.

and resolution, practical, 39–41Distributed Bragg reflector (DBR) diode

laser, 107.Distributed feedback (DFB) diode laser,

107, 113.ultrafast, 113

Dithering vs. halftoning display, 589.DLP. See Digital light processor.DMSO, 697, 726–727, 760, 875.

handling, 739DNA damage, 390, 517, 539, 680, 682–684,

812.DNA probes, 273, 317, 339, 343, 354, 358,

360, 362, 369, 393, 396, 459, 520,531–532, 539–540, 691–695, 774,779, 782, 812, 818–825, 828, 874.

DAPI, 140, 344–345, 355, 358, 376, 431

DRAQ5, 343Hoechst, DNA dye, 136, 339, 344, 360,

362, 520, 565–566, 683, 782DNA sequencing, constructs for, 801–802.DNA transfer, 724, 756, 760, 773, 790,

802–804.Dominant-negative effects, 755.Donor/acceptor pair (FRET), 790, 792–794,

796–797. See also, FRET.before bleach/after bleach ratio, 794equations, 790, 792–794

948 Index

Donor/acceptor pair (FRET) (cont.)fluorescence, 796–797fluorophores, 794separation in nm, table, 793

Double-image, diagram and example, 169.Double-label, 375.Down-conversion, parametric, 114.DPAL. See Diode-pumped alkali lasers.DPL. See Diode-pumped lamp.DPSS. See Diode-pumped solid-state lasers.Drift, 386–387, 652, 655, 732.

CCD read amplifier, 76compensation for, 392–393, 732–733,

886focus, 16, 40, 115, 190, 219, 386, 489,

567, 652, 720, 729, 886compensating, 396, 732

lasers, 85–86, 115DRM. See Digital rights management.Drosophila, 273, 675–676, 747–748,

751–752, 754, 756, 759, 804, 810.living embryo, 675–676, 752salivary chromosomes, 273SPIM, image, 675–676

Duty cycle, laser, defined, 110.DVD, 587–588.Dye lasers, 86, 103, 112, 114, 124, 540–541

in cancer treatment, 112colliding-pulse mode-locked, 112with intra-cavity absorbers, 112noise and drift, 86references, 124as wavelength shifters, 103

Dye-filling, studying micro-cavities,173–174.

Dyes, 22–23, 36, 44, 90–102, 109, 116, 118,165, 173, 183, 212, 222, 342–346,353–358, 360, 430, 461, 462, 527,528, 575, 726, 736–738, 740–745,748, 749, 755, 759–760, 774, 775,782, 804. See also, Green fluorescentprotein (GFP); Rhodamine dyes;Fluorescein.

affect on living cells, 391, 748AlexaFluor, 353–355Aniline Blue, 430–432, 435, 438, 774APSS and Canna yellow, non-linearity,

165bandwidth of emission, 44BODIPY TR, methyl ester, 760BOPIDY, 142, 342–343, 353–355, 389,

692, 749, 760–762Calcein AM, 355, 360, 362–363,

426–427, 430, 685, 804, 812calcium dyes, 346–347cAMP, 347characteristics of probes/specimen, table,

344–345, 354–355coumarin, 114, 339, 344–345, 353, 355,

654–655, 661, 693cyanine, 339, 342, 344, 354–355,

362–363, 374, 443, 540, 587, 760,854, 874

diI derivatives, 355, 362, 389, 726, 760donor acceptor pair, 794. See also, FRETDNA probes, 343–344, 531–532,

818–825, See also, DNA probesDRAQ5, 343dyes vs. probes, 353, for embryos, 748,

761exciting efficiently, 44fade-resistant, 36 See also, Antifade;

Bleachingfor fatty acid, 347. See also, FM4–64,

belowFeulgen-stained DNA, 166, 200, 298,

433, 437Fluo-3 and Fura Red, for calcium, 180,

183, 345, 434Fluo-3 for calcium, 737fluorescein, 353, 355. See Fluorescein;

FITCfluorescence lifetime, 517, 527–528FluoroNanoGold, 854FM4–64, FM1–43, lipophilic dyes, 236,

355, 359–360, 389, 556, 755,760–761

fura-2, 103, 189, 234, 257, 345, 346, 348,358–359, 361, 529, 531, 726–727,730, 733, 741–743, 810, 812, 846,850

Fura Red, 180, 183, 345, 454future developments, 348–349genetically expressed, 348Glutathione, 342, 358, 545, 694, 779,

782hazards in using, 116, 118for ion concentration, 346–347ion-sensitive probes, table, 531kinetics, 741–742lanthanum chelates, 345–346laser/filter configuration, table, 799lineage tracers, 461lipid dyes, 236, 355, 359–360, 389, 556,

755, 760–761living cells, rapid assessment, table, 360loading, uniformity, 749. See also,

LoadingLysoTracker Red DND-99, 360membrane labels, 344–345membrane potential, 205, 346microinjection, 360–361, 388, 739, 748,

755, 795, 803–804MitoTracker Red , 142, 170, 353, 358,

360, 430–431, 692, 750multi-photon excitation, 543–544nano-crystals, 343, 345. See also,

Quantum dotsNile Red, 435, 528, 575, 774, 782organic, 342–343, 353–356oxygen sensor, 347patch clamp loading, 360, 726, 734,

738–740pH indicator, 346, 739–745. See also,

pHimaging

photoactivatable, 187, 210, 224, 383, 385,541, 544–545, 693, 729, 759–760,912

Kaede, 187, 383, 385Kindling, 574, 760PA-GFP, 187, 383, 385, 752, 759–760

photodestruction, 340–341. See also,Bleaching

and Chapter 39photophysical problems, 338–340

absorption spectra, 339autofluorescence, 339–340contaminating background, 339–340optimal intensity, 340Rayleigh/Raman scattering, 339singlet state saturation, 338–339. See

saturation, belowtriplet state saturation, 339

phycobiliproteins, 338, 341, 343,355–357, 693

for plants, 774–775. See Chapters 21 and44

two-photon, 782propidium iodide, 344, 355, 360, 426,

651, 693–695, 773, 778–779, 782,812, 875, 877

quantum yield, 172, 180, 184, 338–845,347, 353–354, 360, 363, 383, 421,543–544, 574, 661, 683, 690–692,710, 737, 792, 794–795

ratio methods, 346–348, 742–743rhodamine, 353, 355. See also,

Rhodamineexcitation, 109

saturation , 21–22, 41, 142, 222, 265,276, 338–340, 448, 643, 647, 899

Schiff-reagent, 262, 369, 770, 774–775,778

selection criteria for, 353–358signal optimization strategies for,

341–342SNARF, 345–346, 531, 739, 744–745specimen damage, 340–341spectral properties, 212, 342, 344–345spectral unmixing, 192, 382, 664–667for STED, table, 575

Dynamic Image Analysis System (DIAS),396–397, 783–784.

living cells of rodent brain, 396of plant cells, 783–784

Dynamic range, 929–930.

Ee2v Technologies, EM-CCDs, 76–77,

233–234, 237, 262, 460, 925–926.E-CARS. See Epi-detected CARS.ECL. See Emitter-coupled logic.Edge detector (software), 309, 322, 327,

396, 823–826.Edge effect, self-shadowing, 172.Edge-emitting diode laser, 89, 106.

corrective optics for, 89cross-section through, 106

Index 949

Efficiency, laser, 102, 105–106. See also,Quantum efficiency (QE); Photonefficiency.

of diode injection lasers, 105–106wall-plug, of argon-ion lasers, 102

EFIC. See Episcopic fluorescence imagecapture.

EGS. See Ethylene glycol-bis-succinimidyl.E-h. See Electron-hole.Electro-magnetic interference, in electro-

optical modulators, 57.Electron microscopy, 167.

brain slices, 730–731chapter, 846–860cryo-techniques, 854fixation, 167, 368–369immuno-stained, 371–372, 852micrographs, 479, 847–853, 855–858tomography (EMT), 610–611

Electron-multiplier CCD (EM-CCD), 30–31,74–75, 78, 142, 233–235, 262,466–467, 482, 647, 678, 737,753–754, 784, 923–926.

advantages and disadvantages, 30–31,220, 228, 233–235, 237, 459–460,647, 737, 909, 923–926

CIC, clock-induced charge, 234, 926and disk-scanners, 76, 205, 215, 220,

270frame-transfer, 262, 234gain-register amplifier, 76–77, 258, 753,

925interline-transfer, 233–234mean-variance curves, 78multiplicative noise, 77noise currents, 256parameters, vs. normal CCD, table, 233QE(effective), 78, 927readout amplifier, 76–77, 258, 753–754,

925results, 235, 237, 755

Electron-beam-scanning television, 6–7.Electron-hole (e-h) pairs and photon

counting, 29.Electronic bandwidth, 64–65. See also,

Bandwidth.Electronic noise, defined, 232.Electronik Laser Systems GmbH, VersaDisc,

109.Electrons, interaction with light, 129–130.Electro-optical modulators (EOM), 25, 54,

57, 87, 116, 543, 701, 903–904.Electroporation, 359–360, 795, 803.

for chromophores, 803Ellis, Gordon, 2, 3, 7, 8, 13, 14, 84, 129,

131, 478, 507.Embryo imaging. See Living embryo

imaging.Embryos, 761–766.

bulk labeling, with dyes, 761depiction, in time and space, 762–764dyes, for multi-wavelength analysis, 756FRET, 764–766

labeled proteins, 756photobleaching, 759transcriptional reporters, 756

EM-CCD. See Electron-multiplier CCD.Emission filter. See Filters.Emission spectra, of arc sources, 136, 176.Emission spectra, fluorophores, 1- vs. 2-

photon excitation, 421.Emitter-coupled logic (ECL), 259.EMT. See Electron microscopy tomography.Endomicroscopy, 511, 513, 514.

distal tip for, 514fiber-optics, 513human cervix image, 513human gastrointestinal track image, 514miniaturized scanning confocal, 511

Endoplasmic reticulum, 374, 770, 819.and DiOC6, 390FLIP, 382FRET, 795genetic fluorescent probes, 771, 783and harmonic signal generation, 703in ion-imaging, 738and phototoxicity, 685

table, 363Endpoint data analysis, 816–817.Endpoint translocation/redistribution assays,

814.Energy diagram, lasers, 102, 105, 106.

argon-ion laser, 102helium-cadmium laser, 105helium-neon laser, 105semiconductor laser, 106titanium:sapphire four-level vibronic

laser, 109Energy, of single photon, 35, 127.Energy transfer rate, for FRET, 790, 792.Entrance aperture. See Back-focal plane.EOM. See Electro-optical modulators.Epi-detected CARS (E-CARS), 597–599.

erythrocyte ghosts, 603Epi-fluorescence microscopy. See

Fluorescence microscopy, 44, 166,172–173, 195, 202, 235.

Epi-illuminating confocal microscope, 9,166. See also, Confocal laserscanning microscopy; Confocalmicroscopy.

Episcopic fluorescence image capture(EFIC), 607–608.

mouse embryo image, 608Epithelial cells, 14–15, 603.

CARS image, 603oral, optical sections, surface ridges,

14–15EPS. See Extracellular polymeric

substances.Erythrocyte ghosts, CARS imaging, 603.Ester-loading technique. See Acetoxymethyl

esters loading method.Ethylene glycol-bis-succinimidyl (EGS),

369.Euphorbia pulcherrima, spectrum, 710.

European Molecular Biology Laboratory(EMBL), 53, 212.

compact confocal camera, 212Evanescent waves, 90, 177, 180, 245, 503,

801.defined, 90, 180optical fibers, 503resolution measurement, 245

Excess light. See Stray light.Excimer lasers, 112, 116.

maintenance, 116for tissue ablation, 112

Excitation efficiency, multi-focal multi-photon microscopy, 552.

Excitation filter, requirements, 44. See also,Filters.

Excitation source, laser. See Lasers; Non-laser sources.

Excitation wavelength change, contrast, 173.Explants, for imaging living embryo,

748–749.Exposure time, 62, 65, 71–76, 81, 127, 137,

141–142, 176, 212, 219, 224, 226,231–236, 267, 270, 276, 346, 363,392–393, 423, 427, 459–460, 477,495, 556, 613, 627–628, 651, 655,681–686, 692–697, 708, 746–747,753–755, 760–764, 783–784, 816,822, 850–851, 873.

for CCDs and EM-CCDs, 127, 137,141–142, 231–236, 267

disk scanners, 231–235laser, safety, 117–118, 839, 900, 903–904reducing, 753–755and source brightness, 141–142total, comparison of methods, 442, 449UV, 116x-ray, 614–616

External laser optics, maintenance, 117.External photoeffect. See Photoemissive

effect.External Pockels cell, 25, 54, 87, 116, 543,

701, 903–904.External-beam prism method, laser control,

90.Extracellular polymeric substances (EPS),

183, 311, 358, 376, 703–704, 717,760, 783, 870, 879–880. See also,Collagen.

bleaching, 693damage, 685dye, 361lectin-binding in biofilms, 870, 879–880matrix, 760negative contrast, 173in optical projection tomography, 612plants, 438, 783preparation, 376

Extrinsic noise, reduction, 21.

FFabry-Perot interferometer, optical cavity,

81–82.

950 Index

Fast Fourier transform, 487.to identify interference fringes, 202

Fast line scanner, 231–232.Fatty acid indicator, 347.FBG. See Fiber Bragg Grating.FBR. See Fiber Bragg Reflector.FBTC. See Fused biconical taper couplers.F-CARS. See Forward-detected CARS.FCS. See Fluorescence correlation

spectroscopy.Feedback, 136, 139.

for control of light-emitting diode, 139to increase source stability, 136

Femtosecond pulsed lasers. See Ultrafastlasers.

Feulgen-staining, DNA, 166, 200, 298, 433,437

Fianium-New Optics, Ltd., FemtoMaster-1060

fiber laser, 113–114.Fiber Bragg Grating (FBG), laser

stabilization, 87.Fiber Bragg Reflector (FBR), stabilizes

laser, 87.Fiber lasers, 85, 101, 109–110, 113–114,

124.defined, 109–110temperature sensitivity, 85tutorial reference, 124ultrafast, 101, 113–114

Fiber optics. See Chapter 26.beam-splitters, 503–504Bow-tie, pol-preserving fiber, 503cable, for delivering ultrafast pulses, 88laser output, 106pigtail, 106

Fiber optics used in microscopy, 501–507.evanescent waves in optical fibers, 503fiber image transfer bundles, 504–505fiber-optic beam-splitters, 503–504fused biconical taper couplers, 503–504glass made from gas, 501gradient-index optical fibers, 501–502key functions of fibers, 505–507

delivering light, 505–506detection aperture, 506diffuse illumination, 507for femtosecond laser pulses, 507large-area detection, 507large-core fibers, as source/detection

apertures, 507same fiber for source and detection,

506single-mode fiber launch, 505SMPP optical arrangement, 216

managing insertion losses, 506angle polishing of fiber tips, 506anti-reflection coating of fiber tips, 506index matching of fiber tips, 506

microstructure fibers, 504modes in optical fibers, 502polarization effects in optical fibers, 503polarization-maintaining fibers, 503

step-index vs. gradient index, 502step-index optical fibers, 501–502transmission losses in silica glass, 502

Fiber-optic confocal microscopy, 501–515,893.

benchtop scanning microscopes, 507–508clinical endomicroscopy, 513

distal tip, 514human cervix image, 513human gastrointestinal track image,

514image transfer bundles, 504–505

managing insertion losses, 506miniaturized scanning confocal, 508–512

bundle imagers for in vivo studies, 509with coherent imaging bundles,

508–509imaging heads, 508–512objective lens systems, 509optical efficiency, 509optical schema, 508resolution, 509rigid endoscope, 511vibrating lens and fiber, 510–511in vivo imaging in animals, 510–514

Fiber-optic interferometer, 240–241, 504,609.

diagram, 241for measuring point spread function,

240–241Fiber-optic light scrambler, 8, 13, 131–132,

143.Fibroblasts, 292, 361, 691, 798, 803, 852.Field diaphragm, 34–35, 127–128, 139, 461,

627, 648–649.Field effect transistor (FET) CCD amplifier,

30–31, 77, 922–927, 929.noise vs. pixel dwell time, 922

Filament-based lamps, 34, 44, 126–132,135–138, 346, 507, 648, 663.

fiber optic, 507image, 100 W halogen bulb, 135size, 126–127spectrum, 44, 136stability, 34, 137

File formats, multi-dimensional images,288–289.

Fill factor.of CCD, 920–921, 927, 929disk-scanning microscopes, 224–228, 233,

552Filtering, digital, 281, 810. See also,

Deconvolution.Gaussian, 41, 65. See also, Gaussian

filtersmulti-dimensional microscopy display,

281nonlinear, deconvolution, 190sets, for automated confocal imaging, 810smoothing, effect on contrast, 59

to reduce “noise” features, 70Filters, optical, 43–51, 70, 89, 162, 190,

212, 753. See also, Heat filters.

conventional, 45hard vs. soft coatings, 45–49intensity loss, 212interference, 45–51

conventional and hard coatings, 46multi-channel detection, 51ND filters, 43, 89notch and edge, 50

tuning with angular dependence, 50to select image contrast features, 162short-pass, interference type, 46transmission vs. laser line, 212types, 46wavelength selective, 43–51

FiRender, 281–282.First or front intensity, projection rule, 302,

304.FITC. See Fluorescein isothiocyanate.Fixation, specimen, 368, 378, 428, 852, 854,

856.antibody screening with glutaraldehyde

fix, 377artifacts, 195, 369–373, 428, 624, 815,

854, 857autofluorescence, 358, 663borohydride to reduce autofluorescence,

374, 770chapter 368–378characteristics, 368–370

chemical fixatives, 369crosslinking fixatives, 369freeze substitution, 369, 769, 854–856microwave fixation, 369protein coagulation, 369

cryo-fixation, 854dehydration, 166, 368, 417–418, 481,

611, 623–624, 815, 849, 854–855effect on plants, 428for electron microscopy, 167, 368–369,

372, 479, 731, 851–860ethylene glycol-bis-succinimidyl, 369evaluation, 371–374

cell height to measure shrinkage,371–373

MDCK cell example, 372, 373formaldehyde, 369–370, 373general notes, 374–378geometrical distortion, 372–373, 815GFP, 854, See also, Green fluorescent

proteinarsenical derivatives, 348

glutaraldehyde, 369, 370high-content screening, 815immunofluorescence staining, 371, 372,

852improper mounting, 376microwave, 377–378mounting methods, 370–374

critical evaluation, 371–374media refractive index, table, 377technique, 371

optical properties of plants, 428pH shift/formaldehyde, 370–371, 373

Index 951

plants. See also, Botanical specimens,Plant cells, 428, 769–770, 773–774

refractive index of mounting media, table,377

optical effects, 428refractive index of tissue/organs, table,

377shrinkage, 369–373, 624, 815, 854staining, 370–371tissue preparation, 376

Fixed wavelength lasers, table, 119–120.Fixed-pattern noise, 74–76, 278, 924, 927,

931.Flare, out-of-focus light, 6, 132, 157–158,

172, 395, 456, 465–466, 469, 471,481, 649, 731.

Flatness of field, 145, 151, 154, 418, 457,639.

measurement/ small pinholes, 145, 457,639

objectives, to improve, 151–152Flat-fielding CCD data, 76, 477.

black reference, 76white-reference, 76

Flexible scanning, 51–52.FLIM. See Fluorescence lifetime imaging

microscopy.FLIP. See Fluorescence loss in

photobleaching.Flip mirrors, to control laser, 58.Floppy disks, 586.Fluorescein,48, 80–81, 88, 203, 261,

353–355, 375, 443, 582, 697, 781,794, 930.

arsenical derivatives, 348calculating laser power needed, 80–81,

443derivatization, diagram, 354double-labeling, 375filters for, 48photobleaching quantum yield, 363rhodamine and, FRET between, 794

Fluorescein isothiocyanate, 88, 198, 203,261, 263, 335, 375, 394, 397–398,511–512, 527–528, 582–583,693–694, 781, 794, 799, 884, 885.See also, Fluorescein.

2-photon, 781biofilms, 884–885dextran, 292, 512filter sets, 48–49FRET, 794, 799lifetime, 527–528, 532photobleaching quantum yield, 363toxicity, 391, 693–694

Fluorescence anisotropy measurements, 742.Fluorescence contrast, 172–173.Fluorescence correlation spectroscopy

(FCS), 5, 363, 383, 385, 602, 801,803, 805, 917.

and CARS, 602FRET, 801

laser requirements, 81table, 385

Fluorescence emission, botanical specimens,425–428.

1- vs. 2-photon excitation, 421Fluorescence imaging, deconvolution vs.

confocal, 459–460, 644–648.Fluorescence in situ hybridization (FISH),

316–317, 319, 323, 331, 333–334,343, 875–878.

biofilms stains, 875–878with fluorescent protein, 878

Fluorescence ion measurement, 736–738,740–745. See also, Calcium imaging,pH, etc.

calcium imaging, 736–737concentration calibration, 742–745indicator choice, 738interpretation, 740–741pH imaging, 346, 739–745water-immersion objectives, 737

Fluorescence lifetime imaging microscopy(FLIM), 108, 111, 114, 139, 204,233, 382–383, 385, 516–533,799–801.

advantages, 766, 800alternatives to, 766analysis, 251applications, 516–518, 527–532calcium imaging, 529chemical environment probe, 517FRET, 517–518ion concentration, 517, 528–530multi-labeling with dyes, 517, 527–528pH imaging, 529–530probes, 517table, 530–532

comparison of methods, 523–527acquisition time, 525–526bleaching, 524cost, 526detector properties, 526–527multi-exponential lifetime, 523–524photon economy, 524–525pile-up effect on detection efficiency,

526shortest lifetime, 523table, 526

decay process of excited molecule, table,518

frequency domain methods, 518–520disk-scanning implementations, 520phase fluorometry method, 518–519point-scanning implementations, 520widefield, spinning-disk, 519–520

frequency-domain, 108reducing repetition rate, 111

FRET, 799–801history, 516Jablonski diagram, 516, 517, 697, 792with light-emitting diode sources, 139limitations, 800living cell images, 204

methods, 518–527comparison, 523–527frequency domain, 518–520time domain, 520–523

multi-focal multi-photon microscopy,555–556

quantitative fluorescence, 517–518quantum efficiency, 516spectroscopy, 516table, 385time domain detection methods, 520–523

point-scanning, 522streak camera, 520TCSPC FLIM, 522–523time-gated FLIM, 523

use of intensified CCDs for, 233Fluorescence loss in photobleaching (FLIP),

187, 382, 384, 801.FRET, affected by, 801table, 384

Fluorescence microscopy, 4, 9. 13, 43–44,154, 166, 172–173, 195, 202, 235,251, 448–451, 809–810 See also,Widefield (WF) fluorescencemicroscopy.

chromatic correction, 154compared to disk-scanning microscopes,

235vs. confocal imaging, 13depth of field, 4filters for selecting wavelengths for,

43–44folded optical path, 166increase contrast with less intensity,

172–173signal-to-noise ratio comparative,

448–451bleaching-limited performance,

448–450configurations of microscope, 448, 449disk-scanning microscope, 449line illumination microscope, 449saturation-limited performance, 450scanning speed effects, 450–451S/N ratios, table, 450wide field (WF) microscope, 450

spectral problems, 44Fluorescence, quenched by colloidal gold,

854.Fluorescence recovery after photobleaching

(FRAP), 51, 54, 56, 90, 187, 210,218, 224, 229, 237, 362, 382, 384,390, 691, 759, 801, 805, 850.

in biofilms, 874damage to cellular structure, 341,

859–851damage to microtubules, 341, 850–851efficiency of illumination light path, 210related to TEM of same specimen,

850–851setups for, 218, 907table, 384using CARV2 disk-scanner, 229, 907

952 Index

Fluorescence resonance energy transfer(FRET), 26–28, 34, 184–187, 204,218, 221–222, 382, 384, 425,517–518, 556, 650, 691, 741–742,764–766, 788–806, 796–797.

based on protein-protein interactions, 800based sensors, 798–799botanical specimens, 425C. elegans, 766chapter, 778–806

cloning and expression of fluorescentconstructs for, 801–804

donor/acceptor pair, 790, 792–794donor, 796–797

efficiency, 792experimental preparation, 795FCS and, 801FLIM and, 799–801between fluorescein and rhodamine, 794fluorescence lifetime imaging, 517–518fluorescent proteins, 794–795FRAP and, 801. See also, Fluorescence

recovery after photobleaching futureperspectives, 805

induced by cholera toxin transport, 797intramolecular, 765kinetics, 741–742in living cells, 195–186, 204

chapter, 788–806in living embryos, 764–766MMM, 797–798nanobioscopy of protein-protein

interactionsacceptor bleach for, 797–798donor fluorescence for, 796–797measurement methods for, 795sensitized emission of acceptor,

795–796photobleaching, 691practical measurements, 792probes, 221–222quantum dots, 801setups, 218small molecules, 794–795spatial orientation factor, 792–793spectrofluorimetry, 793spectroscopic properties used for, 795standards for, 34table, 384theory, 790–794TIRF and, 801total, measured with widefield, 26–28in transgenic animals, 765wavelength depiction, 793

Fluorescence saturation, singlet-state, 21–22,41, 142, 265, 276, 339, 448, 643,647, 899.

Fluorescence speckle microscopy (FSM),13, 383, 385, 889.

table, 385Fluorescent biosensor, 799, 805.

future, 805mitotic clock measurements, 799

Fluorescent constructs for FRET, 801–802.cloning of fluorescent chimeras, 801–802expression and over-expression, 802functional activity of expressed, 802

Fluorescent dyes. See Dyes; Fluorescentindicators; Fluorescent probes.

Fluorescent efficiency, 34.Fluorescent emission, incoherence, 130.Fluorescent indicators, 346–348, 736–743.

See also, Fluorescent probes, andparticular ions.

binding equation, 740–741buffering, 740calcium imaging, 736–737

calibration, 742–743indicators, 738

cellular introduction, 738–739. See also,Loading

cellular trapping, 738choice, 738concentration, 741–742dialysis, 740free diffusion, 741genetically expressed intracellular, 348

green fluorescent protein, 348ion indicators, 348ligand-binding modules, 348

handling, 739–740inaccurate measurements, 740–741intracellular parameters imaged, 346–348

Ca2+, 346–347cAMP, 347fatty acid, 347ion concentrations, 346–347membrane potentials, 346other ratioing forms, 347–348oxygen, 347pH, 346, 739–745wavelength ratioing, 346

positive pressure, 740selectivity, 743

Fluorescent intensity (IF), TIRF, 180.Fluorescent labels, 342–346, 530–532, 761,

775. See also, Dyes; Fluorescentprobes; Chapters 16–17, and byname of dye.

Fluorescent probes. 353–364, 387–389, 517,530–532, 736–737, 739–740, 755,769, 771, 773, 783, 806, 810, 811.See also, Dyes, Fluorescenceindicators and by name of dye,Chapters 16, 17.

automatic living cell assays, 811bound, 737care, 739–740characteristics, table, 344–345, 354development, 736dye criteria for, 353–358

AlexaFluor dyes, 353–355BOPIDY dyes, 353–355, 749, 760–762coumarin dyes, 353, 355cyanine dyes, 353, 374, 587, 760, 854,

874

dye classes, table, 355dye vs. probes, 353fluorescein, 353, 355. See also,

Fluorescein fluorescent proteins,355–357

GFP, 355–357. See, Green fluorescentprotein indicators of intracellularsate, 346–348

Ca2+ indicators, 346–347proteinmulti-photon excitation, 357–358phycobiliproteins, 355–357probes/specimen characteristics, table,

354quantum dots, 357rhodamine, 342–345. See also,

Rhodamineexcitation, 737, 344–345for fluorescence lifetime imaging, 517,

530–532genetically encoded, for plant imaging,

769, 771, 773, 783. See also,Transcriptional reporters;Transfection agents for high-contentscreening, 810

high specificity/high sensitivity, 806living cell imaging, 387–389

rapid assessment by, table, 360loading methods, 358–360. See also,

Loadingacetoxymethyl esters, 359ATP-gated cation channels, 359ballistic microprojectile delivery, 360,

724–725, 802–803direct permeability, 358–359electroporation, 359–360, 795, 803microinjection, 360–361, 388, 739,

748, 755, 795, 803–804osmotic permeabilization, 359peptide-mediated uptake, 359transient permeabilization, 359whole-cell patch pipet delivery, 360,

726–727, 734, 738–740photoactivatable, 210, 224, 383, 385, 541,

544–545, 693, 759–760, 912Kaede, 187, 383, 385Kindling, 574, 760PA-GFP, 187, 383, 385, 752, 759–760

photobleaching, 362–363. See also,Bleaching

phototoxicity, 363–364 See also,Phototoxicity factors influencing,table, 363

specimen interactions, 361–362cytotoxicity, 362localization, 361–362metabolism, 361–362perturbation, 362

target abundance/autofluorescence,360–361

tissues, 360Fluorescent proteins, 187, 355–357, 739,

794–795.

Index 953

emission change after photodamage, 187

FRET, 794–795genetically engineered variants, 739ion binding regions, 739

Fluorescent lights, stray signal, 201, 632,904.

Fluorescent staining, 371, 393, 438, 774.See also, Dyes; Staining.

immunofluorescence, 371, 372, 852living cells, 393microglia, 319–320, 393–398nuclei of living or dead cells, 393

Fluorite (CaF2), optical to reduce chromaticaberration, 153.

FluoroNanoGold, cryosections, 854.Fluorophores, 44, 338–349, 543–544,

664–667, 748, 794, 799. See also,Dyes, Fluorescent labels.

Flying spot detector for measuring photonefficiency, 34–35.

Flying spot ultraviolet (UV) microscope,6–7.

Fly’s-eye lenses, for diode lasers, 107–108.FM4-64, FM1-43, and other lipophilic

membrane dyes, 236, 355, 359, 360,389, 556, 775, 760–761.

Focal CheckTM beads, 657–659.Focal-plane array detection, 2-photon,

542.Focal shift for mismatched RI, 405,

407–410, 553.defined, 405dependence, 410for glycerol, table, 409for water, table, 409

Focus, 3–4, 13, 36, 197.for confocal microscope, 36displacement, by living cell specimen,

22–23effect of coverslip, 197extended, 9in phase-dependent imaging, 13–14planes, diagram, 27position, confocal microscopy, 651–652

Focused spot. See Point spread function.Folded optics, for trans-illuminated confocal

microscopy, 166.Formaldehyde, 369–370, 373–377, 428,

738.AM-loading releases formaldehyde,

738fixation protocol, 371permeabilization agents for, 375pH shift method, 370–371, 373for plants, 428stock solutions, 370–371

Förster distance, defined, 184, 790, 792,793.

Förster equation, 184, 790, 793.Förster resonance energy transfer. See also,

Fluorescence resonance energytransfer.

Forsterite laser (Cr4+ in MgSiO4), 109,

114, 415, 541, 706, 707–709,712–713.

second/third harmonic generation, 114tunable, 109

Forward-detected CARS(F-CARS),597–599, 603.

erythrocyte ghosts, 603Foundations of confocal LM, chapter, 1–19.Four-dimensional images, 746–749, 752,

761–764.advantageous techniques, 746–747automatic image analysis, 321deconvolution, 495embryogenesis visualization strategies,

761–764living cells, 393of living embryos

cellular viability, 747–748challenges, 762dataset display strategies, 393, 763–764deconvolution, 752for large thick specimen, 746–747photobleaching during, 747–748photodamage during, 746required datasets for, 746–747

multi-photon, 535structured illumination, 482SPIM, 676

Fourier analysis.4Pi microscope, 563, 576analogy with image reconstruction, 69of blind deconvolution, 472–476, 478and convolution, 485–487of image formation, 446, 454, 456–457MRM, 618–620of periodic test specimen, 638–639of short laser pulses, 88, 728SPIM multiview processing, 675–677STED, 574of structured-illumination images, 268,

270–273and wavelet processing, 734

Fourier plane. See Back-focal plane, 201,245, 509.

Fourier space, 270–271.Fourier transform, 201, 202, 271, 487, 489,

490–492, 620.of AC interference in image, 201–202,

651and convolution, 487and deconvolution, 487, 490–492for detecting stray light into detector,

201identifying interference fringes, 202of microtubule TIRF image, 183missing cone problem, 494MRM image formation, 620of point spread function, 489, 490

Fractal compression, 581–582.Frame rate. See also, Speed

in confocal microscopy, 11matching, 838–839

FRAP. See Fluorescence recovery afterphotobleaching.

Free diffusion, of fluorescent indicators,741.

Free-ion concentration, 742.Freeze thawing, 731, 739.Frequency, 52, 65, 82.

laser vs. pumping power, 82of resonant galvanometer, 52of sampling clock, 64

Frequency doubling. See Second harmonicgeneration.

Frequency-resolved optical gating (FROG)for pulse length measurement, 115.

FRET. See Fluorescence resonance energytransfer.

Frustrated total internal reflection, defined,177.

FSM. See Fluorescence speckle microscopy.Full-well of CCD pixel, defined, 75.Full-width half maximum (FWHM)

resolution.4Pi, 562, 567of beams in scanning disk, 554of CARS, 597, 599of confocal performance, 656–657,

661–662of emission wavelength

LED, 136quantum dots, 343

of interference filters, 44laser bandwidth, 93, 95, 100, 101laser pulse length, 109, 112, 507, 537,

538, 902micro-surgery precision, 219, 687multi-photon, 682–683, 901–902objective resolution (PSF), 149, 209, 225,

444–445, 456, 492, 509, 552, 571PMT rise time, 225resolution, with spherical aberration,

407table, 409

SPIM, 675STED, 572, 576–578z-resolution, measured, 194

Fundamental limits, chapter, 20–42.Fungi, 438–439, 624, 782, 870.Fura-2 [calcium ion] indicator dye, 103,

189, 234, 257, 345, 346, 348,358–359, 361, 529, 531, 726–727,730, 733, 741–743, 810, 812, 846,850.

Fused bi-conical taper couplers (FBTC),503–504.

Future, 143–144, 160, 192, 219–220, 234.of EM-CCD with interline transfer, 234of laser-scanning confocal microscopes,

219of non-laser light sources, 143–144spherical-aberration corrector, 15, 147,

151, 192of tunable objective, 160

FWHM. See Full-width half maximum.

954 Index

GGain, 31, 232.

of image intensifier, 232photomultiplier tube, from collisions at

firstdynode, diagram, 31

Gain register, (EM-CCD) 76–78, 233–234.

CCD (CCD), 76–78of electron multiplier-CCD, 233–234

Gain setting, 75, 115.defined, 75effect of bandwidth on, 115

GAL4 genes, 773.Gallium arsenide (GaAs).

diode laser, 107, 111InGaAs photodiode, 707–708LEDs, 133, 138, 143PMT photocathode, 4, 28–29, 232, 252,

255, 263, 464, 527, 931Galvanometer, 11, 25, 36, 40, 51–54, 56, 57,

63, 211, 215, 223, 231–232, 513,543, 552, 558, 599, 651–652, 753,806, 907, 910–911, 914, 931. Seealso, Linear galvanometers.

defined, 52–54distortion, 211electromechanical properties, 40errors, 40in fiber-optic micro-confocal, 513figure, 63line-scanner, 231–232linear, 52, 53, 223measurement, 651–656multi-focal, 554multi-photon, 543resonant, 25, 52–54, 56–57, 223, 447,

510, 539, 543, 552, 558, 910specifications for, 214, 543ultra-precise, 211x-y scanners, 213–215, 223, 651–654,

806, 907, 910–911, 914Gamma, brightness non-linearity, 72–73,

287, 832–833.data projector, 590display, 582–583, 589, 832–833

Gas lasers, 86, 90–105. See also, CW lasers;Pulsed lasers.

continuous wave, 90–105maintenance, 116noise sources, 86pressure, 102

Gating, intensified CCD, 25, 233, 262, 522,555.

Gaussian beam profile, lasers, 80–81, 83–84,108–109, 111, 113, 116, 231, 269,338, 456, 496, 502, 538–539, 554,891.

in CARS, 597converted into line, 231, 916fiber optic, 502, 505, 506filling back-focal plane, 210, 509, 629,

633

“Gaussian-to-flat-wavefront” converter,554

Kerr effect produces self-focusing, 111laser beam profile, 538–539, 554, 597,

635–636noise, 473, 497, 925from optical fiber, 502, 505–506optical tweezers, 89. See also, Laser

trapping spatial filter, 89, 729Gaussian filters, digital, 39, 41, 65, 70, 89,

281, 285, 301, 323, 338, 391–392,399, 497, 499, 510, 650, 667–668,676, 729, 734, 753, 764, 830.

of 3D data to reduce Poisson noise, 39,41, 65, 69–70, 269, 281, 285, 323,391–392, 399, 499, 510, 635–636,650, 667–668, 676, 764, 830

“Gaussian blob,” 635–636and Nyquist reconstruction, 65in presentation displays, 830results, 285, 676, 733, 835–837

Gaussian laser pulses, 536–536, 902.Gaussian noise, 473, 497, 925.Gaussian norm statistical tests, 830, 835,

837.GDD. See Group delay dispersion.Gene gun, 360, 724–724, 730.Geometric contrast, 180–187.Geometric distortion, 6, 23, 36, 39–41, 53,

152, 211, 215–216, 265, 297, 329,372–373, 448, 480, 590, 641,653–654, 741, 835.

kinetic, 741measurement, 651–656projector, 590of specimen preparation, 372–373, 815,

872Gerchberg-Saxton algorithm, deconvolution,

472.GFP. See Green fluorescent protein.Ghost images, from transmission

illuminator, 201–202.GIF (Graphics interchange format), 580.Gires-Tournois interferometer (GTI), to

reduce GVD, 88.Glan-Taylor polarizer, 85, 87, 100, 171.

in single-sided confocal microscope, 171

Glan-Thompson polarizer, attenuator, 85,904.

Glutaraldehyde, fixative, 369, 369–374,377–378, 428, 438, 731, 852.

antibody screening with, 377autofluorescence of, 374, 428, 770fixation protocol, 370stock solutions for, 370

Glutathione (GSH), 342, 358, 545, 694, 779.visualization, in plant cells, 782

Glycerol, immersion/mounting medium,404, 407, 409–410, 435, 563, 654,698, 785.

clearing, 198, 200diffusion in, 698

immersion objective lenses, 412, 563, 567example, 785

mounting media, 371, 373, 375, 377–378,420

RI-mismatch, table, 409, 410Goggles, laser, for eye protection, 118.Gold’s ratio method, 476.Golgi receptor, 374, 389, 556, 564–566,

791.Golgi stain, 107, 283, 298.Gourard shading, 308, 309, 311.Gouy phase shift, 597.Graded index (GRIN) lenses, 84.

in diode lasers, 108Gradient index optical fibers, 501–502.Gradient-weighted distance transform,

323.Graphics interchange format. See GIF.Grating, periodic.

GVD compensator, 88, 504, 538, 686laser tuning, 90, 103, 106–107, 111minimum spacing, 1, 16, 652OCT phase-delay, 609pulse compressor, 113spectral detector, 87, 346, 422, 664,

772structured illumination, 266–267, 273

Gray levels, 71–76.intensity spread function, 74–76printer, 592

Green fluorescent protein (GFP), 90, 174,221–222, 348, 355–357, 429,478–479, 556, 568, 571, 612, 614,625, 675–676, 690, 692, 698–699,724–725, 727, 731, 741, 747–752,755, 756–763, 766, 769–773,781–785, 798–806, 812–815, 820,854–859, 862, 873–875, 877–879,885. See also, Transfection reagents;Transcriptional reporters.

biofilms labeling, 873or CFP molecules, as FRET pair, 798constructs, in embryos, 756EM imaging, brain cells, 731, 854–859FRET, 793–795, 798–803image contrast, 174limitations, 760membrane localized, 749methods with Correlative LM/EM, 854mice, 727photoactivatable, 187, 383, 385, 752,

759–760photobleaching, 690, 692, 698for plant imaging, 424, 429–430,

769–773, 781–785direct visualization, 773genetic fusions, 773, 783genetic marking, 773two-photon excitation, 782–783

protein fusions/cytoskeleton, 773–774,801

tagged proteins, 758TIRF, 90

Index 955

FRET, 794Grey levels, 71–76.

printer, 592GRIN. See Graded index.Ground state depletion (GSD), 573.Group delay dispersion (GDD), 537–538,

543.Group velocity dispersion (GVD), 88, 111,

210, 537, 606, 609, 903.in optical coherence tomography, 609pulse broadening due to, 88, 111, 210,

537–538, 543, 606, 609, 728, 903

GSD. See Ground state depletion.GTI. See Gires-Tournois interferometer.Guinea-pig bladder, calcium sparks, image,

237.GVD. See Group velocity dispersion.Gzip, 580.

HHairs, plant, 431, 434–436, 772.Halftoning vs. dithering, 589.Halogen lamps, 126–127, 132, 136–139,

143, 159, 663.brightness vs. temperature, 136filaments, 132image, 135lifespan, 136power available, 126–127stability plot, 137

Haralick features, 818–820.Hard coatings, for interference filters, 45,

48.Hard copy, 580, 590–594.

photographic systems for, 590–591printers, 591–593

aliasing, 592color images, 592digital, 591–593grey levels, 592ink jet, 593laser, 593posterizing, 591scaling techniques, 592

Harmonic signals, 2, 49, 80, 90, 100, 109,113–114, 162–163, 174, 179–180,188, 243, 361, 414, 428, 535, 545,550, 556, 577, 596–597, 682,703–704, 708–719, 722, 729, 734,894 See also, Second harmonicgeneration; Third harmonicgeneration see Structuredillumination.

chapter, 703–721contrast, 179–180, 188descanned detection, 56in lasers, 109, 113, 114, 115plants, 428second and higher, 114

Haze, from out-of-focus light, 227.HBO-50 mercury-arc bulb, 126.HCS. See High content screening.

Heat, 84–85, 89–90, 109, 129, 133.filtering, dichroic filters, 43–44, 129,

132heat sink for LED light source, 133from laser cooling, 84–85, 109of optical trap, 89–90placing system components, 129

Heat filters, to exclude IR light, 43–44, 129,132.

liquid, 132Heating. See also, Thermal variables.

detectors, 252microwave fixation, 377

in magnetic resonance imaging, 621–622multi-focal, multi-photon, 551, 556, 685,

903specimen, by the chamber, 387–389, 394,

732specimen, by the illumination, 43, 89,

132, 211, 218, 341, 536, 539, 544,556, 621–622, 681, 685, 884, 903

calculation, 89, 685, 904stability, 652

HeLa cells, 391–392, 693, 799, 812, 814,820, 828, 854.

Helios Gene Gun System, 724.Helium-cadmium (He-Cd) laser, 83, 86, 90,

93, 103, 105, 115.operational lifetime, 115output variation, 86transverse electromagnetic mode, 83

Helium-neon (He-Ne) laser, 82, 84, 88–90,93, 102–103, 105, 107, 240, 241,376, 673, 680, 798, 799, 864, 875.

four state, 82, 105Heterectis crispa, 874.Hidden-object removal, 304–305.High content screening (HCS), 809–817.

for cytomics chapter, 809–817data management/image informatics,

816–817fluorescence analysis of cells, table, 812multiple fluorescent probes, 810

High resolution spatial discrimination, 813.High throughput screening (HTS), 809.High voltage electron microscope (HVEM),

846.stereo images of platelets, 848–849

Hippocampal brain slices, 268, 316–317,393, 556–557, 722, 724–725, 727.

calcium imaging, 556–557culture protocol, 724–725damage, 341at neurons, 205, 268, 316–317, 393

Histology, 623, 624.Historic overview of biological LM, table,

2–3.Hoechst, DNA dye, 136, 339, 344, 360, 362,

520, 565–566, 683, 782, 812.4Pi, image, 565–566FLIM image, 521high-content screening, 812, 814

Holey optical fiber/non-linear effects, 88.

Holographic diffusers, to reduce coherence,84.

Holography, holomicrography, 7–8.Hooke, Robert, image of cork, 769–770,

785.HTS. See High throughput screening.Huffman encoding, 580–581.Human endomicroscopy, confocal.

cervix, 513gastrointestinal track, 514

Human retina, viewed with OCT, 609.Huygens, 3D software, 104, 413, 669, 778.Huygens-Fresnel wavefront construction,

406.HVEM. See High-voltage electron

microscope.Hybrid mode-locked dye laser, 540–541.Hymenocallis speciosa, fluorescence spectra,

422.Hysteresis.

in Piezoelectric scanners, 57, 754temperature cycling of lenses, 249

II5M, (Incoherent Illumination Image

Interference Imaging), 275, 561,569–570, 672.

optical transfer function (OTF), 569–570ICNIRP. See International Commission of

Non-Ionizing Radiation Protection.ICTM. See Iterative constrained Tikhonov-

Miller algorithm.IEC. See International Electrotechnical

Commission.IF. See Fluorescent intensity.Illumination, 44, 210. See also, Structured-

illumination microscopy, andChapter 6.

brightness, table, 140errors, 211–212evaluating, 211–217goal in confocal microscopy, 210path, 211–212types of lamps, 44vignetting caused by beam shift, 211–212

Image(s), 9, 11–12, 30–31, 38–39, 59, 145,192, 210, 219, 280, 286–290. Seealso, Multidimensional microscopyimages.

contrast, 7, 11, 16, 39, 49, 60–62, 68,159, 162, 165, 167, 173–175, 180,189–190, 192, 201–204, 248, 421,473, 488, 542, 599–600, 607, 622,656, 657, 675

chapter, 162flare, 649

definition, 280degradation of, measuring, 145extended-focus, 9motion between specimen and objective,

39multi-dimensional microscopy, 286–290

anisotropic sampling, 287

956 Index

Image(s) (cont.)calibrating image data, 286–288contrast transfer function (CTF), 61.

See CTFdata type/precision in computations,

288–289digitization, defined, 62dimensions, 286–288display devices, non-linearity of, 72–73file formats, table, 288–289processor performance, 289–290Voxel rendering speed, 290

real, disk- and line-scanners, 30–31reconstructing, and noise reduction,

38–39. See also, Reconstruction;Nyquist reconstruction sharpness ofvs. signal intensity, 192

of source and detector pinholes, 210speed of acquisition, 11–12. See also,

Speed as sum of point images, 59thermal distortion, 219. See also, Thermal

variablesImage analysis. See Automated 3D image

analysis methods; Automatedinterpretation of subcellular locationpattern.

Image dissector, 254–255.in trans-illumination mode, 10

Image enhancement. See Deconvolution,488–499.

Image iconoscope, for television, 6–7.Image intensifiers, 13, 232–233, 235, 255,

460, 477, 519–520, 522, 524,555–556, 730, 737, 784, 801, 930.

Image Pro Plus, 282, 290.Image processing. See also, Automated 3D

analysis methods, and Multi-dimensional microscopy display.

for display, Chapter 14for measurement, Chapter 15

Image resolution, 8, 9. See also, Resolution.Image substrate, automated confocal, 810.ImageJ, free software, 282, 290, 395,

732–733, 762–764, 795, 858.Imaging system, optics characterized by

CTF, 61.Imaging techniques, 382–386, 394–395.

combining fluorescence with other,383–386

fluorescence correlation spectroscopy, 383

fluorescence lifetime (FLIM), 382,516–532

fluorescence loss in photobleaching(FLIP), 382

fluorescence recovery afterphotobleaching, 382

fluorescence resonance energy transfer,382

fluorescence speckle microscopy (FSM),383

laser trapping, 383linear unmixing, 192, 382, 664–667

multi-channel time-lapse fluorescence,382

optical tweezers, 383photoactivation, 187, 224, 383, 385, 541,

544–545, 693, 759photo-uncaging, 383. See also, Photo-

uncaging physiological fluorescence,383

spectral, 382table, 384–385time-lapse fluorescence, 382

Imaris, software, 193, 281–282, 284,287–288, 290–291, 299, 301–303,308, 311–312, 764, 795.

In vitro fertilization, mitotic apparatus, 188.In vitro preparations.

2D mixed-cell, assays, 813antifade agents. See also, Antifade, 342automated analysis, 318–320backscattered light image, 513biofilms, 870, 872, 879, 884bleaching, 551, 851brain slices. See Brain slices, 392–393,

725cell maintenance, 387cytoskeleton, 368fertilization, 188GFP, 357high content screening, 809, 813–816high speed imaging, 11, 237, 809, 813,

815–816ion imaging, calibration, 742living cell imaging, 387micro-CT, 614, 617micro-MRI, 618, 621, 623–625multi-photon, 535optical coherence tomography image, 609photodamage, 684

In vivo (intact animal) imaging, 112,368–377, 512, 545, 806.

2-photon microscopy (MPM), 535, 543,545

cell preparations, 387comparison with fixed material, 368–377FLIM calibration, 517labeling, 372–373miniaturized confocal, 504, 508, 511–513micro-CT, 614, 617micro MRI, 618, 621, 623–625molecular imaging, 806photodamage, 684, 693–694, 698“stick” lenses, 806

Incandescent lamps, 34, 126, 133–137, 477,499 See also, Halogen lamps.

black-body radiation emitted by, 135–136spectrum vs. temperature, 137stability, 137, 477

Incidence angle, 49, 50.efficiency, 143interference filters/transmission, 49reflectivity, diagram, 50

Incident light beam, sample interaction,162–163.

Indo-1, calcium indicator, 103, 189, 257,345, 346, 348, 529, 531, 544, 693,697, 742–743.

Infinity corrected optics, 155–157, 166, 239,405.

advantages, 156–157, 166, 239, 405Infinity PhotoOptical, InFocus spherical

aberration corrector, 15, 151.Infinity space, generating, 157.Information, 27, 60, 64, 73–74, 179, 235,

241, 243, 268, 270–275, 278, 330,334, 353, 369, 382–383, 396, 398,443, 448, 459, 468, 475–476, 481,487, 488–490, 494, 496–499, 506,512–513, 517, 519, 522–524,543–544, 556, 559, 570, 580–587,596, 643, 650, 732, 715, 769, 774,776, 779, 782, 790, 794, 800.

3-dimensional, 321, 378, 396, 7474Pi, 570and bleaching, 222, 690–692, 705CARS, 597–598, 602colocalization, 668confocal, 461, 462contrast, see Chapter 8 and Contrastcrystal orientation, 179, 188display of, 280–281, 288–291, 293,

295–297, 299–301, 304–305, 311efficiency, 336, 628, 631of electronic signal, limitations on, 64genetic, 756, 762–763lost signal, 25–28matching gray levels to, 73–74micro-CT, 615micro MRI, 618and Nyquist sampling. See Nyquist

sampling, 38, 39, 634–637chapter, 59–79

optical projection tomography, 612out-of-focus light, 27, 368, 458, 461, 746,

784parallel vs. serial acquisition, 223–224PSF, 245, 247, 250from second harmonic generation signal,

179Shannon theory, 443on source brightness, 137spectral, 665–667SPIM, 614, 675–378storage, 106

chapter, 580–594theory, 4, 64, 443transmission, contrast transfer function,

37, 60Index mismatch. See Spherical aberration.Infrared (IR) lasers, 89, 383, 385. See also,

Ultrashort lasers; Laser tweezers.solid state lasers, 108–109

Infrared paper, to identify infrared beams forsafety purposes, 118.

Ink jet printers, 593.Innova Sabre/frequency-doubling crystal,

102.

Index 957

Insect cuticle, transparency to NIR light,166.

Installation requirements, for laser sources,85.

Instrument dark noise, 660. See also, NoiseIntegrated circuit (IC) chip, 9.

Intelligent imaging innovations, (III), 3Dimaging system supplier, 78–79, 151,192, 395, 411, 654.

Intensified CCD, 13, 232–233, 460, 477,519–522, 524, 555, 556, 737, 784,930.

Intensity, light, 26, 37, 43, 58, 59, 61,71–72, 86, 87, 133, 136, 163, 165,180, 189, 192, 208, 217, 222, 228,258, 270, 391, 413, 426, 459, 461,487, 536, 538, 571–573, 633, 681,693, 705, 810, 901.

of excitation light, 80, 222, 680–682laser beam, stability, 86losses

detection path, table, 217illumination path, table, 217

minimum needed, 392on optical response of specimen, 165in photons/second, 80regulating, 43, 88singlet-state saturation, See Saturationand visibility, 37

Intensity control.continuous wave laser, 88non-laser, 128

Intensity distribution, 146–154.of Airy disk, 65, 146. See also, Airy disks

changes with focus, 147, 407, 455,463, 471

effect of coverslip thickness, 149effect of RI mismatch, 148. See also,

Spherical aberrationin focal spot, plots, 147–154nonsymmetrical change with focus,

148unit image, 147

with astigmatism, 152with coma present, 151with spherical aberration, 148–150,

212Intensity loss, with spherical aberration in

detection path, 148–150, 212.See Spherical aberration.

Intensity spread function (ISF), 74–78.CCDs and PMTs compared, table, 78defined, 75estimating intensity measurement error,

76and gray levels, 74–75measuring, 75

Interference contrast.differential interference contrast, (DIC),

10, 14, 76, 127, 146, 171, 453, 468,473–475, 846, See also, Differentialinterference contrast.

deconvolution of, 473–475

phase-contrast, 9, 171, 368, 372, 453,506, 643, 649, 731, 851, 854, 890,892. See also, Phase contrast

centering the phase rings, 643. Seealso, Bertrand lens scanning, 9, 13

using fiber optics, 506RI inhomomogeneity and contrast, 22–23,

41Interference filters, 45–51, 102, 136, 212.

in argon-ion laser systems, 102continuously-graded, 137destructive and constructive reflections,

45transmission, 212types, 46–49

Interference fringes, coverslip surface, 168,170.

Amoeba plasma membrane/coverslip, 170in close proximity, 168

Interference mirrors, 46.Interference mode, coherent light, 130.Interference, speckle pattern, 8, 13, 84, 90,

130–132, 144.in backscattered light images, 448fluorescence speckle microscopy (FSM),

13, 383, 385, 889Interferometer.

4Pi microscopy, 561Fabry-Perot (laser), 81–82fiber-optic, for testing objectives,

240–241Gires-Tournois, 88Mach-Zender, to measure pupil function,

245optical coherence tomography, (OCT),

504, 609Twyman-Green, 239

Inter-fluorophore distance, measurement,184. See also, Fluorescenceresonance energy transfer.

Interfocal crosstalk, 227–228.disk scanners, 227–228, 444, 449time multiplexing as solution to,

553–554Interlocks, laser safety, 118.Intermediate optical systems, LSCMs,

chapter, 207–220.Internal focusing elements, in objective,

157, 511.International Commission of Non-Ionizing

Radiation Protection (ICNIRP), 117.International Electrotechnical Commission

(IEC), 117.International television standards, 589.Internet sources. See Links.

lasers, 123, 124Intrinsic noise, 21. See also, Poisson noise.Inverse filter algorithm, 476, 477.Ion-binding in Aequorin emits light, 737.Ion concentrations, 346–347, 517, 528–530,

741.chapter, 736–745determination, 517, 528–530

Ion-concentration imaging, 736–738,740–745. See also, Calcium imaging,pH, etc.

calcium imaging, 736–737concentration calibration, 742–745indicator choice, 738interpretation, 740–741pH imaging, 739–745water-immersion objectives, 737

Ion sensitive probes, optical, 348, 737.table, 531–532

IR. See Infrared; Near infrared.Irradiance, arc and halogen light sources,

130.table comparing, 130

ISO standard, microscope dimensions, 156.Iso-intensity surface, or arc sources, 304.Iterative constrained algorithms, 475–476.

See also, Deconvolution; Nonlinearconstrained iterative deconvolutionalgorithms.

Iterative constrained Tikhonov-Milleralgorithm (ICTM), 497.

JJablonski energy diagrams, 516, 517, 697,

792.Jansson-van Cittert algorithm, 476, 496.Jitter, defined, for scanners, 54.JND. See Just noticeable difference.Joint Photographic Experts Group. See

JPEG.JPEG (Joint Photographic Experts Group),

581–584.Just noticeable difference (JND), ocular

response, 72–73.

KKaede, photoactivatable fluorescent protein,

emission change after photodamage,187, 383, 385.

example image, 187Kalman averaging, 21, 39, 53, 304, 306,

627, 638, 655, 750, 754, 781.comparison with deconvolution, in

reducingintensity, 39

Kepler, Johannes, 788.Kerr cell, 516.

mode-locking (KLM), 111, 133of titanium:sapphire lasing rod, 113

Kerr effect, defined, 111, 179.self-focusing of pulsed laser light, 111

Kindling proteins, 574, 760.Kinetics, 691, 694–698, 741–742, 774, 796,

810–812, 816–817.bleaching, 691, 694–698and endpoint data analysis, 816–817fluorescence, 262–263, 348, 383, 385,

571, 578, 741–742. See also, FLIMFRET, 796high content screening, 810–812,

816–817

958 Index

Kinetics (cont.)ion concentration dyes, 741and STED, 571, 578

Kino, Gordon, confocal design, 6.KLM. See Kerr lens mode-locking.Köhler illumination, 34, 127–128, 131, 229,

251, 627, 648–649.coherence of light, 131in disk scanner, 229field diaphragm, 35, 127–129, 139, 461,

627, 645, 648–649to limit non-uniformity of illumination,

127–128to measure photon efficiency, 34

Krypton laser, 102, 119, 346, 355.comparison with argon-ion laser, 102wavelength, 102

Krypton/argon (Kr/Ar) laser, 90, 92, 93,102, 108, 119, 203–204, 343, 375,748, 798, 811.

stabilization, 88KTP. See Potassium titanium oxide

phosphate.

LLabeled structures, plants, 757, 761, 775.

bulk labeling, living embryos, 761cell walls, 775selective labeling, 757

Label-free microscopy, noise, 114.Lamp housing, 134.Lamprey.

labeled axons, 235, 236larva, optical projection tomography

image, 612Landmark-based registration synthesis

method, 328–329.Lanthanide chelates, 345–346.Large mode area photonic crystal fiber

(LMAPCF), 110.Larmor frequency, MRM imaging,

618–622.Laser(s), 7–9, 44, 80–83, 88, 90, 94,

112–114, 119–120, 131, 540–543,599–600. See also, Fiber lasers;Mode-locked lasers; Multi-photonultrafast lasers; Up-conversion fiberlasers; Ultrafast lasers.

Alexandrite, 109amplifier rods, 116attenuation of, 85, 87–88, 354, 415, 904axial or longitudinal modes, 83basic operation, 81–83, 116CARS microscopy requirements, 599–600chapter, 80–125, table, 119–120coherence, spatial and temporal, 83–84colliding-pulse mode-locked (CPM), 540for confocal, 7, 9–10, 77–78, 280,

535–545continuous-wave, 90–110control of power, 543Cr:Forsterite, 109, 114, 415, 541,

706–709, 712–714

excitation wavelength choice, 540–542.See also, Acousto-optical devices,filters

femtosecond pulsed laser, 44. See also,Ultrafast lasers

fiber-based lasers, 109–111, 113–118table, 94ultrafast, 113–114up-conversion fiber lasers, 109–110

fiber light delivery, 107,See also, Fiber-optics

GaAs, 107, 111gas, 90, 91–10. See also, lasers by gas.

alkali-vapor, 103Ar-ion, 90, 101–102Kr-ion, 102HeNe, 102–103HeCd, 103

heat removal, 84hybrid mode-locked dye laser, 540–541important properties for confocal, 80light delivery, 87–89

fiber-optic, 106mirrors, 88

longitudinal modes, 82–83maintenance, 115–116

active media replacement, 115cooling components, 116–117optical resonator, 116

metal vapor, 112microscopical uses

nonlinear: 2- 3-photon, 90Raman and CARS, 90TIRF, 90tweezers, 89. See Laser trapping

multi-photon. See Multi-photonmicroscopy

Nd:glass, 706–708Nd:YAG, lasers, 88–89, 91, 95, 97, 103,

107–109, 111, 113–115, 117, 218,245, 514, 680, 798

Nd:YLF, lasers, 89, 98, 100, 103, 109,112–114, 750, 760–761

Nd:YVO4, lasers, 89, 95, 100, 103,107–109, 111, 113–114, 541

NO SMOKING, 116performance tables, 91–101phase randomization, 8, 13, 131–132, 143pointing error, 87

active cavity stabilization, 87polarization, 83, 88–89power control, 543pulse broadening/compensation, 88,

901–904pulsed, 110–115. See also, Titanium-

sapphire, Cr:Forsterite,Nd:glass,YAG/YLF/YVO4, etc.

cavity dumped, 111Kerr lens mode-locked, 111modulated diode lasers, 112pulse-length measurement, 115,

901–903purpose, 110

saturable Bragg reflector, 111ultrafast, DPSS lasers, 112ultrafast, fiber lasers, 113white-light continuum lasers, 113why are they useful?, 110

pumping power requirements, 82safety, 117–118, 839, 900. See also,

Safetygoggles, 118screens and curtains,118, 904

solid state, 103. See also, Solid-statelasers

semi-conductor, 105–107thin-disk lasers, 109

spectrum of light, 44stabilization, 85–87

active, 87titanium:sapphire laser, 82, 84–86, 88–91,

94, 100–103, 105, 107, 109,111–112, 114, 123–124, 165, 346,358, 415, 423–424, 459, 541, 550,551, 645–647, 688, 706–708, 713,727, 750, 756, 759

4Pi, 563–564, 567brain slices, 731CARS, 599compared to other fast lasers, 82–83,

85, 110, 112–113embryos, 731, 750, 756, 759, 764maintenance, 116and OPO, 114–115plants, 415, 423–424, 706–708,

713–714, 717, 781–783popular models, specs, table, 120STED, 575

transverse modes, 82–83, 85, 110tweezers, 89. See Laser trappingtypes, 90ultrafast fiber, 113–114, See also,

Ultrafast laserswavelength expansion by sum-and-

difference mixing, 114optical parametric oscillators, 114–115second/third harmonic generation, 114

white light continuum lasers, 88, 109, 113continuum, 88, 109He:Cd, 113.

Laser cavity stabilization, active, 87.Laser cutters, 686–687.

integration, 218–219Laser illumination, conditions for, 8.Laser lines, using acousto-optical tunable

filters, 56.Laser media, maintenance, 115–116.Laser printers, 593.Laser rods, maintenance, 116.Laser Safety Officer, 117.Laser sources, 9, 80–125. See also, Lasers.Laser speckle, 84, 90, 130–132, 448.

removing, 84. See also, Scramblerssource, 130

Laser trapping, 80, 89, 110, 218–219, 383,385, 539, 646, 680.

Index 959

Laser tubes, operational lifetime, 102, 115.components likely to fail, 115

Laser tweezers. See Laser trapping.LaserPix, 282.Laser flying-spot microscope, 7.Lasersharp, confocal microscopes, 282, 284,

285, 288, 292, 296, 302–306.LaserVox, 281–282.Lateral chromatic aberration (LCA), 14,

155–156, 239, 242–243, 287, 640,657–658.

correction in conventional optics, 155measured, 657–658

Lateral coherence, 8, 84, 267.Lateral resolution, 1–4, 9, 11–13, 28, 207,

209, 222, 225, 230, 238, 270, 320,409, 453, 511, 513, 542, 552, 554,563, 568, 651, 654–656, 747. Seealso, Resolution.

4Pi, 568CARS, 596–597, 599confocal endoscopy, 511, 513confocal optics, improvement, 9, 651,

654–656of display, 292light microscopy, 1–3optical coherence tomography, 609–610with pinhole and slit disks, 225and spherical aberration, 409SPIM, 613, 674STED, 573–575, 578table, 209, 409

Laterally-modulated excitation microscopy,see Stuctured-illumination.

LCA. See Lateral chromatic aberration.LCD. See Liquid crystal display.LCOS. See Liquid-crystal-on-silicon.LCS (Leica Microsystems AG), 282, 312,

910.Lecithin myelin figures, CARS image, 204.LED. See Light-emitting diode.Leica, confocal manufacturer, 51–53, 56–57,

160, 218, 797, 910.acousto-optical beam-splitter, 160, 218objective lens transmission, 160RS Scanner, 52–53spectral confocal, TCS SP2, 51, 56–57,

910tube length conventions, 157, 239

Leica Microsystems AG, 282, 910.Leica TCS 4Pi, 119–120, 565–568.

4Pi microscopy type C, 565–568imaging of living cells, 568lateral scanning, 567, 910mitochondrial network image, 568optical transfer function (OTF), 567sketch, 566, 910thermal fluctuations minimized, 567

Lempel-Ziv-Welch (LZW), 580–582, 584.Lens aberrations, 13–15. See also,

Aberrations.Lens focal length, change, with wavelength,

152.

Leonardo da Vinci, early optical studies,788–790.

Leukocytes, 347, 387, 520, 815, 854.automatic analysis, 815multi-photon, phase-based FLIM, 521

Lifetime. See Fluorescence lifetime imagingmicroscopy.

Ligand-binding modules, 256, 348, 741,846.

Light detection, general, 28–33, 251–264.See also, Detectors; specificdetectors: CCDs, PMTs, etc.

assessment of devices, 260–262charge-coupled device (CCD), 254comparison, table, 233, 255–256, 647conversion techniques, 259–260direct effects, 252future developments, 262–264history, 262–264image dissector, 254–255microchannel plate, 232–233, 255, 262

gated, 519, 523–524, 527, 532MCP-CCD, 262

noise internal to, 256–259internal detection, 256noise currents table, 256photoemissive devices, 256–257photon flux, 257–258pixel value representation, 258–259

photoconductivity, 252, 253photoemissive, 254photon interactions, 252–256

work functions, table, 252–253photovoltaic effect, 252–253point detectors, 260–261. See also, PMTquantal nature of light, 251–252thermal effects, 252vacuum avalanche photodiode, 254, 255

Light dose, related to pixel/raster size, 64.Light, effects, on plant cells, 770. See also,

Bleaching, Phototoxicity.Light-emitting diode (LED), 34, 54,

132–133, 135–139, 143, 237.aligning, 135control by current-stabilized supply,

138–139definition, 105to detect galvanometer rotor position, 54excitation wavelength for fluorophores,

136expected cost reduction, 237fluorescence image, 142galvanometer position feedback, 53lifespan, 137to measure photon efficiency, 34microscope illumination, 131–139, 141,

143organic, projected development, 143radiance, 138spectra, 133stability, 136temperature effects, 137wavelength vs. current change, 137

Light flux, light-emitting diode temperature,133.

Light intensity, 71, 163.Light microscopy history, 1–4.Light paths. See also, Commercial confocal

light microscopes.separating excitation/emission, 44–45

Light piping by specimen vs. depth, 182.Light-sheet illumination, 672–673.Light sheet microscopy, 613.

chapter, 672–679optical setup for, 613white-light continuum lasers, 113

Light sources, widefield, 132–139, 143. Seealso, Chapters 5 and 6, Arc lamps,LEDs, Lasers; Nonlaser lightsources; Filaments; Halogen.

commercial sources, 143solar, 126–127, 131, 135stand-alone, 143table, comparative performance, 140types, 132–139

Light transmission, 11, 139, 160–161,223–229.

cummulative loss along optical path, 139of Nipkow disk system, 11, 223–229specifications for objectives, table,

160–161Lighting models, 3D image display,

306–312.absorption, 309–312advanced reflection models, 309artificial lighting, 309–312Gourard shading, 308gradient reflection models for voxel

objects, 309Phong shading, 308–309Phong/Blinn models, 308simulated fluorescence process, 310surface shading, 310transparency, 280, 284, 287, 300, 304,

309, 311–312Lilium longiflorum, image, 783.Limitations, confocal microscopy, chapter,

20–42.fundamental, 20–42table, 41, 647typical problem, 21, 24

Linear galvanometers, 54.Linear longitudinal chromatic dispersion

(LLCD), stereoscopic confocalimage, 154.

Linear unmixing. See Spectral unmixing.Line-scanning confocal microscope, 50, 51,

231–232, 237, 784, 908, 916.Linearity, 72, 490.

deconvolution for image enhancement,490

display advantages and disadvantages, 72Links (Internet addresses).

2 photon excitation spectra, 546, 727,729, 782

brain slices, 727

960 Index

Links (Internet addresses) (cont.)CCDs, 76, 234, 927, 931components, 58confocal Listserve, 390, 901deconvolution, 495dyes, 221, 343–344, 782fluorescent beads, 653FRET technique, 185, 803high-content screening systems, 811image management, 865lasers, 104, 115, 120, 123–125live-cell chambers, 388–389, 870movies related to book, 235, 392muscles, 237non-laser light sources, 138, 143plants, 769safety, 900software, 282, 376, 594, 734, 762, 764,

776, 777, 820, 824, 827, 831–833,844, 845, 864–862, 865–867, 869

SPIM, 672Lipid dyes, 236, 355, 359–360, 389, 556,

755, 760–761.Lipid receptors, 790.Liquid crystal-on-silicon (LCOS), 266.Liquid crystal display (LCD), 39, 67, 73,

291, 293, 589–590.digital projectors, 590filters, 928non-linearities, 73shutters, 299, 929supertwisted nematic (STN), 589thin-film transistor (TFT), 589

Liquid crystal technology/dynamicpolarization microscopy, 188. Seealso, Pol-scope.

Lissajous pattern, circular scanning. 554.“tornado” mode, SIM scanner, 52

List servers, 125.Lithium triborate (LBO), as non-linear

crystal for multiplying infraredoutput, 109, 115.

Living cells, 80, 90, 114, 136, 145–161,167, 219, 221–222, 381–399,429–439, 480, 564–566, 568,746–766, 770, 772–773, 788–806,811, 813. See also, Brain slices,Plants cell imaging, and bycell/organism name.

2-photon, penetration, 749–7512D plus time, 753–754, 762–7643D projection, 7634D data, 746–747, 7644Pi microscopy, 564–565, 568acquisition speed, 222, 753–754algorithms, 763–764assays, 811beauty and functionality, 790bleaching of, 797. See Bleaching;

Photodamagecell-chamber, 11, 22, 191, 219, 370–371,

386–387, 394, 429–430, 564,610–611

for 4Pi confocal, 564for biofilms, 870–873, 875, 877, 880,

885for brain slices, 394, 723, 727, 729for epithelial cells, 370–371, 377, 386finder chamber, 683flow chamber, 870–873, 875, 877, 880,

885for high-content screening, 810for optical projection tomography,

610–611perfusion, 394for plant cells, 191, 429–430simple, 22, 394for SPIM, 613, 625, 673table of required functions, 380table of suppliers, 388–389test chamber/dye, 654, 661

cell-cycle effects, 790chromatin, 385, 390–392, 684, 693–695,

812chromatin dynamics, 390–392CNS tissue slice preparation, 393confocal microscopy, 381–399, 746, 813

difficulties, 381future directions, 398–399

considerations, 386–390antioxidants, 390experimental variables, table, 386fluorescent probes, 387–389maintenance of cells/tissues, 387minimizing photodynamic damage,

136, 389photon efficiency, 141–161, 389–390in vitro preparations, see In Vitroin vivo preparations, see In Vivo

contrast, 747dyes, 748. See also, Dyes; Fluorophors

etc.for rapid assessment, table, 360

embryos, imaging, 746–766. See also,Living embryo imaging

external membranes, SHC image, 90fluorescent staining, 393

microglia, 393nuclei, living/dead cell, 393

fluorophore effects, 748FRET imaging, chapter, 788–806future, 221–222handling data, 395–396imaging techniques, 382–386, 394–395low-dose imaging, 391–392microglial cell behavior example,

392–398no damage from SHG imaging, 114online confocal community, 390photon efficiency, 141–161, 389–390phototoxicity, 390–391

assays for, 813plant, 429–439. See also, Plant cell

imaging reflectance imaging, 167second harmonic generation. See also,

SHG

of external membranes, 90no damage, 114

test specimen for, 390widefield, 646–647, 751–753working distance, 5, 9, 129, 145, 154,

157, 198, 249, 511, 568, 598, 634,673, 678, 727–728, 747, 774, 779,781, 872

table, 158Living embryo imaging, 749–751, 762–764.

aberrations caused by, 747apparatus, 748C. elegans, 746, 748deconvolution helps confocal, 751–753developmental changes, 746Drosophila, 273, 675–676, 747–748,

751–752, 754, 756, 759, 804, 810dyes, 748

introduction of, 755embryo size vs. speed acquisition,

753–754explants, 748–749future developments, 766fluorescent probefour dimensional, 746–747, 749

cellular viability, 747–748challenges, 762dataset display strategies, 761–764photodamage during, 746–748

high speed acquisitiondisk-scanning confocal microscopy,

754hardware, 754–755

light scattering, 747optimal acquisition, parameters, 753–754refractile specimens, 747superficial optical sections, 748thick specimens

effective strategies, 748–753, 755–761inherent trade-offs, 747–748selective plane illumination (SPIM),

751“Test drives,” for living embryo imaging,

752.widefield/deconvolution, 751–752

LLCD. See Longitudinal chromaticdispersion.

LMA-PCF. See Large mode area photoniccrystal fiber.

Loading methods, fluorescent probe, 347,358–360, 430, 732–734, 738, 739.

acetoxymethyl esters, 359, 360. See also,Acetoxymethyl esters

ATP-gated cation channels, 359ballistic microprojectile delivery, 360,

726, 803direct permeability, 358–359electroporation, 359–360, 795, 803ion indicators, 738–739, 742low level, 430membrane permeant esters, 359–360microinjection, 360, 361, 388, 739, 748,

755, 795, 803–804

Index 961

neurons, 722, 726, 730, 732–734osmotic permeabilization, 359peptide-mediated uptake, 359plant cells, 769stabilizing chemicals, 341–342, 362transient permeabilization, 359whole-cell patch pipet, 360

Local projections, display, 305–306, 307.Location proteomics, 818.Longitudinal chromatic aberration, 152–155.Longitudinal coherence length, 7, 8, 84,

130, 131.Longitudinal linear chromatic dispersion

(LLCD) objectives for 3D color-coded BSL confocal, 154.

Long-pass filters, 43–44.Low-voltage scanning electron microscope

(LVSEM), 846–847, 849–850, 852.LSM. See Laser-scanning confocal

microscopes; Laser-scanning flying-spot microscope. 6–7

Lucoszs formulation, 273.Luminescent nanocrystals, 343, 345.Luminous intensity vs, color, dye molecule,

138.LVSEM, 846–847, 849–850, 852.LysoTracker Red DND-99, 359–360,

709–710.rapid assessment table, 360spectra, 710

LZW compression. See Lempel-Ziv-Welch.

MMach-Zehnder interferometry, 245.Machine learning. See Automated

interpretation of subcellular patterns.Macrography, 3D light scanning, 672.Magnesium fluoride (MgF2).

for anti-reflection coating, 158Magnetic disks, 586.Magnetic resonance imaging (MRI), 618.Magnetic resonance microscopy (MRM),

618–624.amplitude modulation for RF carrier, 620applications, 623–624

botanical imaging, 624developmental biology, 624histology, 623phenotyping, 623

basic principles, 618–619Fourier transform/image formation, 620future development, 624hardware configuration, 621–622image contrast, 622–623image formation, 619–621Larmor frequency, 620Schematic diagram, 618–619strengths/limitations, 622

Magnification, 24, 35–41, 62, 131, 215, 443.See also, Nyquist sampling; Over-sampling; Undersampling.

calibrating, 653, 658and CCD pixel-size, 62, 70

confocal, 52–53, 62–64effect on pixel size, 24, 928factor, 24, 28and lateral chromatic aberration, 278for line-scanner, 232over-sampling, 68–70, 493, 509, 635, 729

high-content screening, 816and pinhole size, 28under-sampling, 68zoom magnification, 11, 24, 37, 63–34,

66, 70, 79, 317, 389, 493, 627,634–636, 731

Maintenance.cell viability, 387dye lasers, 114lasers, 115–117, 124remote logging of, 864troubleshooting reference, 124

Maize (Zea mays), 167–168, 172, 179, 202,417–424, 428, 438, 710–711,713–714.

2-photon, time-lapse microspectroscopy,423

abnormal vasculature, 437anther, 420, 433attenuation spectrum, leaf, 418cross-sections, stem, 172, 707emission spectrum, 710, 711, 713fluorescence spectra, 422–424leaf,

attenuation spectrum, 418optical section, 172, 179reflectance, 167surface, 436

meristem, 420, 430–432, 707multi-photon excited signals, 422–424polarization microscopy, 707, 711pollen grain, 202, 433–434protoplast, 424root, 432second harmonic imaging, 707, 711silica cells, 428, 437, 707spectrum, 422, 423, 710starch, 420, 435–436, 707, 711stem

attenuation spectra, 417, 418, 713optical sections, 419, 714

storage structures, 420, 435–436, 707,711

Manufacturers. See also, Commercialconfocal light microscopes;Appendix 2.

listing with web addresses, table,104–105.

Mapping conventions, in image processing,294–296, 300–304.

data values, 300–304choosing data objects, 300–301object segmentation, 301–302projection rules, 302–304scan conversion, 301–302table, 300visualization, 300

multi-dimensional image display,294–296

G function, 294image/space view, 296orthoscopic view, 294reducing geometric dimensions, 294rotations, 294–296visualization process, 294

MAR. See Mark/area ratio.Marching cubes algorithm, 301–302, 304,

776.Marconi, CAM-65 electron multiplier CCD

camera, 76. See also, EM-CCD.Mark/area ratio (MAR), 279.Marsilea quadrifolia, 416, 419.

attenuation spectra, 416optical section, 419

Mass balancing, to reduce scanner vibration,54.

Mass storage, 580–588, 593–594.data compression for, 288–289, 292–293,

295, 319, 499, 580–585. See also,Data compression

algorithms, 319, 580archiving systems, 580color images, 581file formats, 580–588

removable storage media, 585–588. Seealso, Removable storage media

random-access devices, 586–588sequential devices, 585–586solid state devices, 588

time required, table, 581Materials, silicon, fused quartz, beryllium,

52.Mathematical formulas, for confocal

microscope performance, table, 209.

Maximum intensity projection, 180,284–285, 292, 294, 298, 302–304,307, 313–314, 319, 325–326,330–331, 585, 755, 763–764, 770,774, 881, 884.

local, 305Maximum likelihood estimation (MLE),

472–475, 495, 497–498, 669.blind deconvolution, 472–475, 498, 784effect on colocalization, table, 669

M-CARS. See Multiplex CARSmicrospectroscopy.

MCP. See Microchannel plate, 232–233,255, 262.

MCP-CCD, 262Gated intensified, 519, 523–524, 527,

532MCP-PMT. See Microchannel plate

photomultiplier.MDCK cell, 372–374.

actin cytoskeleton, 374Golgi apparatus, image, 374morphologic changes, 374stereo image, 373, 374vertical sections, image, 372

962 Index

Measurements, 20, 33–36, 76, 139–141,159.

achromat performance, 194buffering of, ion measurement, 738, 740field flatness, 26–28geometric distortion, 653–654laser pulse length, 109, 112, 115, 507,

537, 538, 902–903light throughput, 139–141limits on confocal intensity, accuracy, 20photon efficiency, 33–36pinhole, effective size, 34intensity spread function histogram,

74–78resolution, 241–245, 657, 658shrinkage, specimen preparation,

371–373spectral transmission of objective, 159spherical aberration, 145, 407surface height, using LLCD BSL

confocal, 224z-resolution, 194

Mechanical scanners, 51–54.Melles Griot catalog, real lens, performance,

210.Membrane permeant esters, 361, 358–359,

361, 726, 738–739, 744.Membrane potentials, 179, 188, 204–205,

346, 353, 383, 517, 743, 811–813.Memory stick, 588.Mercury arc lamp, 37, 44, 132, 135–138.

fluorophores matching excitation,135–136, 139

iso-intensity plots of discharges, 132and pinhole size, 37radiance, improvements, 137–138wavelengths, 44

Mercury-halide arc source, 136, 138,143–144.

spectrum, 144Mercury-iodine (Hg-I) arc lamp, radiance,

138.Mercury-xenon arc lamps, 136–138.

spectral lines, 136Meristem, 168, 420, 430, 432, 770,

776–778, 782.maize, 168, 432

Merit functions, confocal scanners, 217.object-dependent, defined, 217object-independent, defined, 217

Mesophyll cells, 169, 193, 195, 417–418,423, 428, 430, 711–712, 714, 779.

A. thaliana, 193, 196photodamage, 203protoplasts, 196, 203, 424, 425–426,

430, 439harmonic images, 711–712, 714image, 424spectra

attenuation, 416, 418change with 1- vs. 2-photon, 421, 423emission, 423

Metal-halide light source, 136, 143–144,907, 908.

Metal vapor lasers, 112.Metamorph, 281–282, 290, 311, 817.Microchannel plate (MCP) image intensifier,

233, 255, 519, 532.multiplicative noise, 233photocathodes, 262PMT, 255, 523, 532

Microchannel plate PMT (MCP-PMT), 255.Micro-computerized tomography (Micro-

CT), 614–618.contrast/dose, 614–615dose vs. resolution, graph, 616layout, 614mouse images, 615–617

tumor-bearing, 617operating principle, 614

Micro-CT. See Micro-computerizedtomography.

Microdissection.with multi-photon IR light, 686–687with nitrogen lasers, 112

Microelectrodes, for introducing indicator,738.

Microglial cell behavior, 392–398.Microinjection, 360–361, 388, 739, 748,

755, 795, 803–804.of chromophores, 803–804

Microlens array, 12, 134, 135, 216, 225,231, 235.

for 4Pi confocal, 563–565for CCD, 237for disk scanners, 6, 12, 216, 224, 226,

231, 458for light-emitting diode source, 134–135for multi-focal, multi-photon, (MMM),

537, 551–555, 558principle, 135in Yokogawa disk-scanning confocal, 12,

224–226, 231, 235Microscopes, 217, 226. See also, particular

types.attachment of confocal scanner, 217specification comparisons, table, 226

Microscopy laboratory URLs, 125. See also,Links.

Microspectroscopy, 421–425, 426, 516.CARS, 601–602fluorescence properties of plants, 421–425lifetime, 516of maize, 424multi-photon setup, 424

Microspores, birefringence images, 189,431–432.

Microsporogenesis, 431–432.Microstructure fibers, 504.Microsurgery, 112, 219, 686–687, 764–765.Microtubule, 11, 68, 80, 188, 222, 292, 432,

582, 703, 714, 752–753, 759, 773,790, 852. See also, Cytoskeleton.

birefringence, 714–715Brownian motion of, 11electron microscopy, 848, 850fixation, 369, 372–375fluorescence correlation spectroscopy, 383

GFP, 12. See also, Green fluorescentprotein

in mitosis, 759. See also, Mitoticapparatus

polarization microscopy, 15, 173, 188,420–421

photodamage of, 341, 850–851stabilizing buffers, 852STED, 576–577stereo image, 752TIRF, 180, 183second harmonic generation, for tracking,

90Microwave fixation, 377–378.Microwire polarizer (Moxtec Inc.), 85.Mie scattering, 162–163, 167, 417–418.

clearing with index-matched liquid, 167comparison with Rayleigh scattering,

163light attenuation in plant tissue, 417by refractive structures, 162–163

MII. See Multi-photon intrapulseinterference, 88.

Mineral deposits, plant, 163–420, 436–437,703.

Miniaturized fiber-optic confocalmicroscope, 508–512.

bundle imagers for in vivo studies, 509clinical endoscope, 514objective lens system, 509optical efficiency, 509optical schema, 508resolution, 509rigid endoscope, 511single fiber designs, 510vibrating lens and fiber, 510–511in vivo imaging in animals, 512

Minolta, CS-100 radiospectrometer, 139.Minsky, Marvin, 2, 4–6, 11, 141, 216, 890.Mirror coupling, pulse width and pulse

shape, 88.Mirrors, 26, 48, 54, 63, 209–210, 214.

galvanometer, 54. See also,Galvanometers

internal, testing reflectance losses, 26laser-line, 48performance, 54, 63.scan angle and magnification, 63size calculation for LSCM, 209x-y scanning mirror orientations, 214

Mismatch, 893.probe shape/pixel, 39, 466caused by chromatic aberration, 243refractive index, 377, 404–412, 411, 654,

658, 747, 863, 8934Pi, 568causing signal loss, 148–150, 408–409,

654chapter, 404–412corrections, 411–412embryos, 747film vs. CCD, 590harmonic signal generation, 704–705less, at long wavelength, 416

Index 963

measurement, 148–150, 655–656of movie frame rate, 839MRM, contrast agent vs. imaging timemylar flakes, 198resolution loss measured, 192–194vector mismatch in CARS, 596–597,

600z-distortion, 287

Mitotic apparatus, 15, 173, 373–374, 377,386, 421, 431, 693, 749, 752, 799.See also, Microtubules.

damage, 693fixation, 373–374, 377FRET, 765marker, cyclin-B, 790Pol-scope, 13, 188, 432, 468, 479–480.

deconvolved, 479images, 15, 188, 479, 717

SHG imaging, 702, 718–719in vitro fertilization, 188

Mitotracker stain, 142, 170, 353, 358, 360,430–431, 692, 750.

living cells rapid assessment, table, 360Mixing, sum or difference, to generate laser

wavelengths, 114.MLE. See Maximum likelihood estimation.MMM. See Multi-focal, multi-photon

microscopy.MMM-4Pi microscopy, 556.MO (magneto-optical) disks, 586.Model-based object merging, 323–325.Mode-locked lasers, 87, 101, 111–114, 118,

124, 358, 520, 646, 728–729, 749,901–904.

active, pulsed laser class, 111adjustment of, 901–904for CARS, 599–600colliding pulse, 112fiber, ytterbium and neodymium,

113–114fiber/diode, ultrafast, 113–114FLIM, 520interference with, by specimen, 171Kerr lens, 111modulator, fiber lasers, 111multi-photon, 535–536, 540–541,

550–551, 563–564, 567, 646,728–729, 749

passive, 111, 113–114saturable Bragg reflector, 111SHG, THG, 706–707

Mode-locked oscillators. See also, Mode-locked lasers.

nanojoule pulse energies, 111Moiré effects, 270–271, 755.

ambient fluorescent room lighting, 201banding patterns, 755disk-scanners and CCDs, 231, 754–755structured-illumination methods, 268–271,

273Molecular imaging, in vivo, 387, 618, 624,

790, 803–806.FRET, 790, 803–806micro-CT, 618

MRM, 624Monitors, computer display, 588–589.Monkey cells, 693, 803.Monomeric red fluorescent protein (mRFP)

constructs, 756, 760, 798.with CFP or GFP molecules, as FRET

pair, 798Montage synthesis method, 281, 312, 318,

328–331, 748, 753, 851–852, 855,858–859.

defined, 329–330examples, 330–332, 780–781scanning electron micrographs, 851–852,

855TEM methods, 858–859

Moon, early phase measurements, 788, 789.Morphological filters, 285, 300–301,

316–317, 320–322, 730–734, 817,826.

high-content screening, 812, 819, 826Morphometry, 145, 316, 319, 331, 726, 728.

group properties, 331intensity/spectral measures, 331interest points, 331invariants, 331location/pose, 331shape measures, 331size measures, 331texture measures, 331topological measures, 331

Mosaicing. See Montage synthesis method.Mounting medium, 166, 198, 342, 370–371,

373–377, 404–413, 418, 454, 457,473, 493–794, 499, 564, 631, 642,652, 655, 696, 730, 774 See also,Clearing agents.

brain slices, 730chapter, 404–413clearing solutions, 166, 417–418, 420,

439, 610, 624, 774–775effect of glass bead, 199plant specimens, 418, 431, 774refractive index, tables, 377selection, 198, 631

Mouse, 192, 376, 393, 608, 612, 615+, 723,726.

confocal colonoscopy, 509, 512embryo

optical projection tomography image,612

SIM/EFIC image, 608GFP transgenic, 726hippocampus, 393micro-CT image, 614–615, 617

femur, 616tumor, 617

spectral unmixing image, 192, 382,664–667

examples, 665–666visual cortex brain slices image, 723

Movement contrast, 190.Movie compression, 836–840.Moving-coil actuators, galvanometer, 52.Moxtec Inc., Microwire polarizer, 85.

MPA. See Multi-photon absorption.MPE. See Multi-photon excitation.MPEG display formats, 836–841.MPEM. See Multi-photon microscopes.MPLSM. See Multi-photon laser-scanning

microscopy.MPM. See Multi-photon microscopy.MQW. See Multiple quantum wells.mRFP. See Monomeric red fluorescent

protein.MRI. See Magnetic resonance imaging.MRM. See Magnetic resonance microscopy.Multi-channel experiments, 813.

filters and dispersive elements, 51time-lapse fluorescence imaging, 382,

384toxicity, 755

Multi-dimensional microscopy, display,280–314. See also, Automated 3Dimage analysis methods.

2D pixel display space, 291efficient use, 292

animations, 292–293artificial lighting, 306–308CLSM images, 286–290

anisotropic sampling, 287calibrating image data, 286data type/computational precision,

288–289dimensions available, 286–287file formats for

calibration/interpretation, 288–289image data, 286image size available, 287image space calibration, 287–288image/view dimension parameters,

table, 288processor performance, 289–290storing image data, 286voxel rendering speed, 290

color display space, 291–292commercial systems, tables

available systems, 282–283desirable features, 288–289display options, 293geometric transformations, 295projection options, 300realistic visualization techniques,

307criteria for choosing visualization, 281data values, definition, 222, 280dimensions, 280, 323degrees of freedom, optical image, 8–9depth-weighting, 304, 306

exponential, 304linear, 304recursive, 304

display view, definition, 280hidden-object removal, 304–305

local projections, 305–307z-buffering, 304–305

highlighting previously defined structures,284

image, definition, 280

964 Index

Multi-dimensional microscopy, display(cont.)

image/view display options, table, 293geometric transformations, table, 295

intensity calibration, 304iso-intensity surface, 304

laser-scanning microscopy, 280lighting models, 306–312

absorption, 309–312advanced reflection models, 309artificial lighting, 309–312Gourard shading, 308–309gradient reflection models/voxel

objects, 309Phong shading, 308–309Phong/Blinn models, 308simulated fluorescence process, 310surface shading, 310transparency, 309–312

living cells of rodent brain, 392–398mapping data values, 300–304

choosing data objects, 300–301object segmentation, 302projection rules, 302–304scan conversion, 301–302segmenting data objects, 301visualization model, 300

mapping into display space, 294–296G function, 294image/space view, 296orthoscopic view, 294reducing geometric dimensions, 294rotations, 294–296visualization process, 294

measurement capabilities See also,Chapter 15

reconstructed views, 312–313results, 284–285

objective vs. subjective visualization, 281prefiltering, 281principle uses, 281–285projection/compositing rules, 302–304

alpha blend, 302, 304average intensity, 302first or front intensity, 302Kalman average, 304maximum intensity, 302

pseudo color, 173–175, 190, 291purpose, 281–285, 293–295realism added to view, 306–308

techniques for, table, 307reconstructed view generation, 290–312

5D image display space, 291–294. Seealso, 5D image display spacechoosing image view, 291–294

subregion loading, 290–291reconstruction, definition, 280reflection models, 306–308rendering, definition for, 280software packages, table, 282–283stereoscopic display, 293, 296–299

color space partitioning, 297interlaced fields of frame, 297

pixel-shift/rotation stereo, 297stereo images example, 298synchronizing display, 297

true color, 291unknown structure identification, 281–284viewing data from, 283visualization parameters, table, 285z-coordinate rules, 304z-information retained by, 296–300

non-orthoscopic views, 299stereoscopic views, 296–299temporal coding, 299–300z-depth, 299–300

Multi-fluorescence, systems for utilizing,217+.

Multi-focal, multi-photon microscopy(MMM), 221, 276, 550–559, 797.

4Pi-MMM, 563–564basics, 565scheme, 563

alternative realizations, 554–555background, 550beam subdivision approaches, table, 558current developments, 558–559experimental realization, 551–555FRET, 797imaging applications, 556

boar sperm cells, 557Convallaria, 556FRET, 556hippocampal brain slices, 557pollen grains, 556Prionium, 556

interfocal crosstalk, 553–554, 556time-multiplexing, 553–555

limitations, 556–558localization, 538Lissajous pattern of scanning foci, 554

“tornado” mode, SIM scanner, 52Nipkow-type microlens array, 551–552optimum degree of parallelization,

550–551resolution, 552–553schematic diagram, 552time multiplexing, 553–554variants, 555–556

FLIM, 555–556MMM-4Pi, 556SHG, 556space multiplexing, 555

Multi-length fiber scrambler, 8. See also,Scramblers, light.

Multi-photon absorption (MPA), 535.Multi-photon excitation (MPE), 356–358,

535–545, 894. See also, Multi-focalmulti-photon microscopy.

absorption, 705–707advantages/disadvantages, 644–647,

749–751autofluorescence, plants, 424, 427background from SHG/THG, 361,

708–709, 728backscattered light imaging, 429

bleaching, 218, 338, 539–540, 680–689,692–693, 905. See also, Bleaching;Chapter 38

caged compounds, 187, 383, 543–544,692, 729, 912

cell viability during imaging, 544–545chromophores for, 543–544detection, 538duty cycle, 644excitation localization, 538excitation spectra, 125FLIM, 576fluorophores for, 543–544FRET, 797heating, 539–540history, 535image formation, 535–540instrumentation, 540–543, 900–905. See

also, lasers for. See also, Ultrafastlasers

Alexandrite, 109Cr:Forsterite, 109, 114, 415, 541,

706–709, 712–714Nd:glass, 706–708Nd:YAG, 88–89, 107–109, 514, 680,

798Nd:YLF, 89, 112–114, 750, 760–761Nd:YVO4, 89, 95, 107–109, 113–114,

541Ti:Sapph. See Laser, titanium-sapphire

lasermulti-focal, multi-photon microscopy

alignment, 900–901beam delivery requirements, 541control of laser power, 543CPM laser, 540descanned detection, 166, 208, 212,

428, 537, 540–542excitation wavelengths, 541focal plane array detection, 542hybrid mode-locked dye laser, 540–541lasers/excitation wavelength choice,

540–542non-descanned detection, 185, 201,

218, 381, 447, 456, 507, 542, 552,559, 643, 646, 727, 750, 779, 904,909, 910

non-mechanical scanning, 543optical aberrations, 542power requirements, 541, 903, 904pulse spreading due to GDD, 547, 538,

543resonant scanning, 543whole-area and external detection,

541–542optical pulse length, 537–538

group delay dispersion, 537–538, 543group velocity dispersion, 88, 111, 210,

537, 606, 903measurement, 115, 901–903

penetration, 749–750photodamage, 539–540, 680–688,

692–693

Index 965

physical principles, 535–540refractive index mismatch, 404–413resolution, 539SHG and THG background, 361,

708–709, 728two-photon absorption cross-sections, 125

(URL) 543–544wavelengths, 538–539

Multi-photon intrapulse interference (MII),88.

Multi-photon microscopy (MPM), 10–11,56, 172–177, 210, 535–545, 681,682, 685–688, 746–766, 894,900–905.

advantages/disadvantages, 644–647,749–751

alignment, 901–902autofluorescence, 425–427, 545SHG, THG, 361, 708–709, 728calcium imaging, 545cell damage during, 544–545, 682, 685

1-photon vs. 2-photon excitation, 681absorption spectra of cellular absorbers,

681intracellular chromosome dissection,

688mitochondria, 686nanosurgery, human chromosomes,

686–687by optical breakdown, 198, 680, 682,

685, 687, 703, 705photochemical, 682–685photothermal, 539, 545, 681, 685,

904reproduction affected by ultrashort NIR

pulses, 686ultrastructural modifications, 685–686

cell viability, 544–545compared with other 3D methods,

644–647, 748–751deconvolution, 495–498developmental biology, 545, 746–754,

757, 759–760, 764dispersion as problem, 56. See also,

GVD; GDDfluorescence, contrast, 172–177for living embryo imaging, chapter,

746–766need for efficient illumination light path,

210optical layout, 540photobleaching, 545, 680–688, 692–693practical operation, 900–905protein damage/interactions, 765resolution, 552setup/operation, 540, 900–905

schematic diagram, 540, 901–902in vivo (intact animal) imaging, 545ultrafast lasers, 88, 90, 109. See also,

Ultrafast lasersAlexandrite, 109Cr:Forsterite, 109, 114, 415, 541,

706–709, 712–714

Nd:glass, 706–708Nd:YAG, 88–89, 107–109, 514, 680,

798Nd:YLF, 89, 112–114, 750, 760–761Nd:YVO4, 89, 95, 107–109, 113–114,

541Ti:Sapph. See Laser, titanium-sapphire

laseruncaging, 545

Multi-photon-based photo-ablation, 764.Multi-slit design, for disk-scanning

confocal, 229.Multi-view deconvolution, 330, 675–677.Multiple quantum wells (MQW), diode

injection lasers, 106.Multiplex CARS microspectroscopy (M-

CARS), 601, 602.Multiplicative noise, 28–33, 51, 77–78, 224,

234, 256–258, 262, 275, 443, 460,633, 661, 667.

of EM-CCD, 30–31, 77–78, 264, 256,262

losses in effective QE from, 33, 234, 443

from PMT, 29, 51, 77–78, 233, 256–258,460, 633, 661, 667

and quantum efficiency, 33, 234, 443photon counting, 32–33, 78

pulse pile-up, 32–33, 35, 78, 521, 523,526–527

table, 256why it is usually unnoticed in LSCM,

633, 661Muscle, 737, 739–742.

fatigue, 739–740

NNA. See Numerical aperture.Nanobioscopy, protein/protein interactions,

795–798.acceptor bleach, 797–798donor fluorescence, 796–797FRET measurement, 795sensitized acceptor emission, 795–796

Nanoscale resolution with focused light,571–578. See also, Stimulatedemission depletion (STED)microscopy.

breaking the diffraction barrier, 571–573different approaches, 573–574

ground state depletion (GSD), 573STED, 573–574

outlook, 577RESOLFT concept, 571–573resolution, new limiting equation, 571

measured, 578stimulated emission depletion (STED),

573–578axial resolution increase, 576compared to confocal microscopy, 576dyes, suitable, table, 575OTF comparison, 578PSF comparison, 578

Nanosurgery, 219.with multi-photon systems, 90

NCI60 CMA, standard encapsulation, 816.NCPM. See Non-critical phase matching.ND. See Neutral-density filters.Near infrared (NIR) lasers, 10, 90, 106. See

also, Lasers: titanium-sapphire; Nd:;Cr:Forsterite.

Near infrared (NIR), 10, 90, 106.diode injection lasers, 106for laser tweezers, 90objective lenses designed, 174

Nearest-neighbor deconvolution algorithm,476.

image enhancement, 495–496Negative contrast, for fluorescence

microscopy, 173–174.Negative feedback, to correct mirror motion,

53.Neodymium glass laser, 706–708.Neodymium-yttrium aluminum garnet

(Nd:YAG) lasers, 88–89, 91, 95, 97,103, 107–109, 111, 113–115, 117,245, 514, 680, 798.

infrared range, 108pumping non-linear crystal/green light,

114–115Neodymium-yttrium lithium fluoride

(Nd:YLF) laser, 89, 98, 100, 103,109, 112–114, 750, 760–761.

Neodymium-yttrium orthovanadate(Nd:YVO4) laser, 89, 95, 100, 103,107–109, 111, 113–114, 541.

kits utilizing, 113Nerve cells, images.

Alexa stained, 330backscattered light images, 167eye, optic nerve, 481Golghi-stained, 298Lucifer-yellow, 314microglia, 396–398rat-brain neurons, 398transmitted light, 475

Neutral-density filters (ND), 43, 76, 126.in fixed-pattern noise measurements,

76to reduce source brightness, 43, 126

NFP. See Nominal focal position.Nikon, confocal manufacturer, 13, 15,

119–120, 161, 199, 201, 507,638–640, 657, 750, 910.

C1 confocal microscope, 119–120, 507C1si spectral confocal microscope, 908,

910CF objectives, 154–156, 217, 669, 779

confocal x-z, BSL image, 22Plan Apo objective, 13, 15, 638resolution, measured, 16, 638–640,

657water-immersion lenses, 15

high-content screening, 810tube length conventions, 157, 239

Nile Red, dye, 435, 528, 575, 774, 782

966 Index

Nipkow disk scanning, 2, 5–6, 11, 12, 41,215, 223, 231, 276, 551, 754,783–784, 810, 894. See also,Yokogawa; Disk-scanning confocalmicroscopy.

commercial systems, 907, 913, 915compared to single-beam scanning, 458for high-content screening, 810micro-lens system, 6, 12, 216, 224–226,

231, 234, 237, 551–552multi-photon, 537, 551–558, 563–565.

See also, Multi-focal, multi-photonmicroscopy rotation, 754

for single-sided confocal, 6, 141, 223,229

source brightness, 141speed of image acquisition, 11, 220,

222–226, 227, 231for tandem-scanning, 141, 215visualization, of cells, 458, 667, 754,

784Nipkow, Paul, 5–6, 109NIR. See Near infrared.Nitrogen lasers, 112.

nanosurgery using, 219NLO. See Non-linear optical effects.NMR. See Nuclear magnetic resonance.Noise, 21, 28, 74–77, 83, 87, 114, 190, 232,

256–259, 442–444, 495. See also,Signal-to-noise ratio; Poisson noise;Quantum noise.

background, 443–444of CCD detectors, 30–31, 77–78,

232–233, 256, 262equations, 256table, 256vs. photomultiplier tube detectors, 74,

77CIC, clock-induced charge, EM-CCDs,

234, 926in counting quantum-mechanical events,

21deconvolution reduced noise, 39–40, 114,

392, 495, 498, 667, 783, 835–836detector, 28fixed-pattern, 74, 76, 278, 924, 927, 931in fluorescence microscopy, defining,

74–75in lasers, sources, 85–86

reducing, 87limits grey levels, 443measurement, 74–75multiplicative, 28–33, 51, 77–78, 224,

234, 256–258, 262, 275, 443, 460,633, 661, 667

in photon detectors, 256–259noise currents table, 256photo flux, 257–258photoemissive devices, 256–257pixel value represented, 258–259

Poisson. See Poisson noisepolarization, in laser systems, 83read, and readout speed, 77

shot, 442–443. See also, Poisson noisesingle-pixel, 65, 67, 190, 635, 832,

835–836deconvolving, to reduce, 39–40, 392,

498, 667, 784, 835–836reducing, 39, 40, 190, 41, 65, 392, 498

sources of, 442–444wavelet transform to reduce, 733–734,

819–820Nomarski DIC contrast, 2, 368, 746, 892.

See also, Differential interferencecontrast.

Nominal focal position (NFP), 405, 408,409.

calculations for glycerol, 409calculations for water, 409z-responses, diagram, 408

Non-confocal microscopy vs. confocal, 746.high content screening, table, 811

Non-critical phase matching (NCPM),114–115.

Non-descanned detection, for MPM, 185,201, 218, 381, 447, 456, 507, 542,552, 559, 643, 646, 727, 750, 779,904, 909, 910.

for CARS, 559No-neighbor algorithm, 476–477, 496.Non-laser light sources, chapter, 126–144.

arc sources, 130, 132, 140commercial systems, table, 143comparative performance, table, 140control, 138for disk-scanning confocal, 141filament sources, 135–136LEDs, 132–133, 135, 138–139, 143light scramblers, 131–132measured performance, 139–141results, 142solar, 126–127, 131, 135stability, 136–137

Nonlinear constrained iterativedeconvolution, 68, 458, 475–476,496–497, 499, 520, 568.

Nonlinear conversion, tunable laser, 114.Nonlinear crystals, frequency multiplying,

109.Nonlinear optical (NLO) effects, in

microscopy, 90, 114, 163, 165, 177,179, 188, 190, 195, 416–417,426–427, 430, 442, 504, 535, 507,703–720, 728, 741, 751. See also,Multiphoton/microscopy; Harmonicsignals; SHG, THG.

absorption, 188, 415–418, 426–427, 430,705

bleaching, 536, 550, 558, 645, 680–685,693, 697, 707, 729. See also,Bleaching; Photodamage

CARS, 595–598, 600DIC, 473–474. See also, Differential

interference contrastfluorescence, 172, 179focus shift with spherical aberration, 409

harmonic generation, 704–705emission, 710–711energy state diagram, 705multi-photon absorption/fluorescence,

705second harmonic generation (SHG),

704–705setup, 708–709third harmonic generation (THG), 705

light sources/detectors, 706–708light attenuation spectra in plants, 706photodetector characteristics, 707pulsed-laser, table, 706. See also,

Ultrafast lasersin optical fiber, 504–508optically active animal structures,

714–717man-made collagen matrix, 717signal-producing structures, table, 715spindle apparatus, 717–718zebrafish embryo, 716, 718

optically active plant structures, 710–714Canna, 710Commelina communis, 712emission spectrum of maize, 710, 711maize stem, 711, 714potato, 712rice leaf, 712, 715

polarization dependence of SHG, 717,719

setup for, 708–710spectra, 415, 417, 435

Euphorbia pulcherrima, 710maize leaf, 710Pyrus serotina, 711

STED microscopy, 571–579. See also,STED microscopy.

structured illumination, 270, 276Non-radiative dipole-dipole interactions,

790.Non-specific staining, 27, 44, 74, 303, 345,

357–358, 442, 467, 472, 617, 660,667–668, 760, 820, 878, 882. Seealso, Background.

Non-tunable solid-state laser, 103.Normal, free-running, pulsed laser, 111.Northern Eclipse, software, 282.Notch filter, to transmit laser line, 49.Novalux Inc., Protera 488 laser system, 107.NSDC. See Nipkow spinning-disk confocal.Nuclear import analysis, 802.Nuclear magnetic resonance (NMR), 618.Numerical aperture (NA), 1, 4, 24, 28, 61,

126, 141, 145, 148, 168, 180, 195,198, 239–250.

affects surface reflection contrast, 180defined, 1determining axial resolution, 4, 241–242,

657determining lateral resolution, 1,

241–242, 656diffraction orders accepted by, 61

effect on self-shadowing, 168, 198

Index 967

in fiber-based mini-confocal endoscopes,509

image brightness, 126matching to CCD pixel size, 62, 928objective lenses with high, 145, 239–250

empty aperture, 248with oil-immersion vs. water objective,

148pinhole size as function, 28and refractive index mismatch, 147–148.

See also, Spherical aberration intandem scanning confocalmicroscopy, 141

vertical shadowing, 195and zoom setting optimal, 24

Nyquist criterion, and digitization, 38–39,64–68.

Nyquist digitizing, 65, 67.Nyquist filtering, 70–79, 281.Nyquist frequency, 64, 301. See also,

Shannon sampling frequency.Nyquist, Harry, 64.Nyquist noise, 256.Nyquist reconstruction, limit output

bandwidth, 59, 66–67, 69, 70, 173,235–236, 280–315, 458, 468–469,474–475, 496–497, 563, 585, 603,607, 610, 615, 635, 672, 675,677–678, 690, 772, 730–731, 762,77, 774–776, 778, 784, 883.

Nyquist sampling, 24, 37, 39, 40, 53, 60,64–70, 73, 75–76, 78–79, 142, 146,152, 205, 222, 258, 271, 273, 289,386, 391, 448, 635–636.

blind spots, 38for CCD camera, 70, 233, 273, 928and deconvolution, 59, 65, 67–68,

222–223, 635diagram, 60optimal, results of deviating from, 24practical confocal microscopy, 448,

635–636reconstruction, see Nyquist

reconstruction.relationship with Rayleigh-criterion and

PSF, 39, 60, 64, 66signal-to-noise ratio, 67, 448subpixel, resampling, 478–479

OObject scanners, image quality, 216.Objective lenses, 13, 15, 25–26, 34, 49, 145,

152, 156, 239–250, 652–660. Seealso, Aberrations.

apodization, 250axial chromatic registration, 287, 658axial resolution measurement, 656–657

vs. pinhole size, 656chromatic aberrations 14, 145, 152–156,

160, 177–178, 209, 242–243, 641,659

apparatus in measuring, 243–244, 654,659

axial shift, 243–245, 657–658chromatic registration, 657–658cleaning, 642confocal performance, 145–161, 652–660contrast transfer function (CTF), 16, 35,

37–39, 59–62, 656, 747coverslip thickness, table, 654

dipping lenses, 161, 209, 411, 429, 568,613, 727, 737, 870, 872

dry, high-NA, aberrations, 15field illumination, 34–35, 127–128, 139,

461, 627, 658flatness of field, 145, 151–152, 154, 418,

457, 639, 659Focal CheckTM beads, 657high-NA planapochromat, 13, 145,

239–250infinity correction, 155–157, 166, 239,

405advantages, 49

lateral chromatic registration, 657–658lateral resolution. See CTFlight, vector nature, 267mounting media. See Mounting mediaphoton efficiency losses, 25–26plan objectives, table, 152point spread function of high NA,

239–250measuring, 240–242, 455, 462, 471,

656polarization effects, 249–250pupil function, measured, 245–248

3D point spread function restored,247–248

empty aperture, 248Mach-Zehnder interferometry, 245phase-shifting interferometry, 245Zernike polynomial fit, 245–247table, 247

resolution test slide, 169, 656spherical aberration. See Spherical

aberrationcorrection, 654–655

sub-resolution beads, 181–182, 196, 454,477, 493, 499, 527, 652–656, 784,900, 904, 930

images, 656table of suppliers, 653

temperature variations, 248–249transmission, optical, 154, 158, 159–161.

See Transmission, objectivetable of objective lenses, 159–161

water-immersion, 145, 149–150dipping objectives, 161, 209, 411, 429,

568, 613, 727, 737, 870, 872use and limitations, 15

working distance, 5, 9, 129, 145, 154,157, 198, 249, 511, 568, 598, 643,673, 678, 727–728, 747, 774, 779,781, 872

x-y and z resolution using beads, 656OCT. See Optical coherence tomography.OLED. See Organic light-emitting diodes.

Olympus, confocal manufacturer, 52–53, 54,119–120, 161, 184, 187, 204, 229,230, 234–236, 419, 421, 427, 557,708–709, 727–730, 797, 908, 912.

Fluoview-1000, 119–120, 184, 187, 204,908, 912

DSU disk-scanning confocal microscope,229–230, 234–235, 908, 913

FRAP system, 210FRET, 797high content screening, 811objectives, 557, 727–730

stick, in vivo objectives, 806TIRF objectives, 183transmission, table, 159, 161

SIM scanner, 52–54tube-length conventions, 157, 239

On-axis reflections, artifact, 171.Onion epithelium (Allium cepa), 390.Online confocal community, Listserv,

390.OPA. See Optical parametric amplifiers.OpenLab, 282.Operational lifetime, of laser tubes, 102.OPFOS, Orthogonal-plane fluorescence

sectioning, 672–673.OPO. See Optical parametric oscillators.OPT. See Optical projection tomography.Optical aberrations, 109, 542. See also,

Aberrations.thin-disk laser optics, 109

Optical layout of confocal microscopes,212–213. See also, Optical paths

by class, 213evaluation, 212–213

class 1 systems, 212class 2 systems, 212–213class 3 systems, 213

Optical bandwidth/electronic bandwidth, 32.See also, Bandwidth.

Optical breakdown, 198, 680, 682, 685, 687,703, 705.

Optical coatings, maintenance, 116.Optical coherence tomography (OCT),

609–610.of human retina, 609schematic, 610Xenopus laevis embryo, 610

Optical components, chapter, 43–59.Optical density (OD), 71, 81, 416.

filters, 43, 49–50Optical disks, 586.Optical efficiency, improvements, 143–144,

216. See also, Photon efficiency.of disk scanners, 216of light-emitting diodes, 143–144

Optical elements, 43–58, 128, 211.confocal light beam affected by, 211of Köhler illumination components, 128

light beam characteristics affected by,211

chapter, 43–58

968 Index

Optical excitation, diagram, 82.Optical fiber. See Fiber optics.Optical fiber, for scanning by moving fiber

tip, 213–214.Optical heterogeneity, specimen, 22–23.

reflection, refraction, scattering, 192–197Optical images, electronic transmission, 5–6.Optical materials, 158, 501.

thermal properties, 158, 248–249Optical parametric amplifiers (OPA),

100–101, 112, 114–115, 118, 124.components, 115table, 101

Optical parametric oscillators (OPO),100–102, 111–112, 114–115, 118,541, 600.

for CARS microscopy, 600cavity dumped, to increase white light,

113tunable, 114–115

table, 101Optical path. of.

4Pi, confocal, 563commercial, 566

acousto-optical device, 55compound light microscope, 156–157CARS, 599, 601, 907CARV-2 disk scanner, 230confocal, 10, 208–209, 212, 632, 681

beam-splitter, 213disk-scanner, 12, 216folded, 166scanning systems, 214

fiber-optic confocal, 508interferometers, 243, 245Kino single-sided disk scanner, 229LaVision-Biotec, Trimscope, 907Leica, TCS AOBS, 910magnetic resonance imaging, 621Minsky confocal, 5, 25for measuring photon efficiency, 34multi-photon, 540, 681, 708–709

multi-focal, 552, 555spectrometer, 424

Nikon C1si, 911Olympus DSU disk-scanner, 230Olympus Fluoview-1000, 912optical coherence tomography, 610optical projection tomography (OPT),

611Petran tandem scanner, 228selective plane illumination (SPIM), 613,

673or simultaneous BSL and fluorescence,

128surface 3D imaging, SIM/EFIC, 608surface spherical aberration, 405–406STED, 573structured-illumination, 266Visitech VT-Infinity and VT-eye, 914Yokogwawa dual-disk-scanner, 231,

915Zeiss LSM-510, META, 916–917Zeiss LSM-5-Live, 50, 232, 916

Optical performance, practical tests,652–660.

axial chromatic registration, 658axial resolution using mirror, 656–657chromatic aberration, 659chromatic registration, 657–658contrast transfer function (CTF), 656coverslip thickness vs. RI, table, 654field illumination, 658flatness of field, 659Focal CheckTM beads, 657, 658lateral resolution, 655resolution test slides, 655–656specimen self-lensing artifacts, 659spherical aberration correction, 654–655

Optical power, specimen plane, table, 140,644.

Optical probes, 737. See also, Dyes;Fluorescent indicators; Fluorophors;Fluorescent labels.

Optical projection tomography (OPT),610–613.

lamprey larva, 612mouse embryo, 612refractive index, 613setup, 611

Optical pulse length, 537–538. See also,Pulse broadening.

group delay dispersion, 537–538group velocity dispersion, 537measurement, 115, 901–903

Optical resonator in laser, 81–82, 116.laser, 81–82maintenance, 116

Optical sectioning, 9–10, 13, 180, 182, 222,223, 236, 268–270, 469, 748,763–764, 772, 774, 775, 784. Seealso, Deconvolution, Confocal, etc.

algorithms for widefield, 763–764of A. Thaliana root, 772, 775with confocal laser-scanning microscope,

9–10example, 182, 463, 471, 492, 656

dynamic imaging, 784improvement, with deconvolution, 752latex bead, 3D image, 196limiting excitation, 223near surface of living embryo, 748near to refractive index interface, 180selective plane illumination, 748structured illumination, 268–270with widefield phase-dependent imaging,

13Optical system, losses, 25–32, 217.Optical transfer function (OTF), 164–165,

490–491, 562, 563, 567, 569–570,578. See also, Point-spread function;Contrast transfer function.

4Pi microscopy, 562, 563, 567contrast, 164–165deconvolution for image enhancement,

490–491I5M, 569–570

point spread function, 490–491. See also,Point-spread function

STED comparison, 578Optical tweezers, 89–90, 110, 218, 383, 385.

setups for integrating, 218table, 385trapping wavelength, 89–90

Optics, general, 12, 125, 156–157.finite vs. infinity, 156–157

Optiscan confocal endoscope, 213–214.Organic dyes, 109, 203, 342–343, 353–356.

See also, Dyes; Fluorophores;Fluorescent labels; Fluorescentprobes.

AlexaFluor, 353–355BOPIDY, 353–355classes, table, 355coumarin, 353, 355cyanine, 353–355fluorescein, 353–355rhodamine, 109, 203, 353, 355

Organic light-emitting diodes (OLED), 143.Orthogonal-plane fluorescence sectioning

(OPFOS), 672–673.Oryza sativa. See Rice.Oscillating-fiber scrambler, 8.Osmotic permeabilization, 359.OTF. See Optical transfer function.Out-of-focus light.

deconvolution vs. confocal microscopy,461.

information, 26, 32, 487, 644–646.Output amplifier, reconstructing analog

signal, 64.Output modulation, of semiconductor lasers,

108.Overheating, of filters, 43. See also,

Thermal variables.Overlap alignment protocol, montaging,

732.Over-sampling, 60, 70, 728.

vs. duplicate-and-smooth process, 70reasons for, 68subpixel, resampling, 478–479

Oxygen sensor, 45, 347.

PPack-and-go mode, Power Point, 842, 844.Paeonia suffruticosa, 421.Panda pattern, polarization-preserving fiber,

88.PAS. See Periodic-acid Schiff.Passively mode-locked lasers, 111.Patch clamp, for loading dye, 360, 726–727,

734, 738–740.Patch pipette, 738.Pattern analysis. See Automated

interpretation of subcellular patterns.Patterned-illumination microscopy, see

Structured illumination microscopyPC. See Personal computer.PCA. See Principal component analysis.P-CARS, Polarization-sensitive detection

CARS.

Index 969

PCF. See Photonic crystal fiber.PE. See Photoelectrons.Pear (Pyrus serotina), spectrum, image,

711.Pearson’s correlation coefficient, 668.Pellicle beam-splitter, 216, 228–229, 231,

346.Peltier cooling.

CCDs, 234, 447cell chamber, 387–389lasers, 85, 106–108, 111, 117

Penetration depth, 177, 343, 643, 672, 731,765.

of dyes, 360, 387, 731, 739, 882, 874of fixative, 369–370, 376, 857FRET sensors, 798–799long laser wavelengths, 109, 416, 418,

427–428multi-photon, 381, 418, 433, 435, 439,

543, 545, 558, 646, 684, 708, 714,728, 749, 904

in plant imaging, 779in scanning electron microscopy, 847in SPIM, 613, 675–678TIRF, 177–178

Peony flower, autofluorescent petals,173–174, 176, 421, 423.

Peptide-mediated uptake, 359.Perfusion.

chambers, 381, 386–389, 394, 726, 729,769, 870–873

fixation, 376Periodic grating. See Grating.Periodic-acid Schiff (PAS) reagent, 262,

369, 770, 774–775, 778.maize pollen grain, 202

Periodically poled (PP) waveguides,114–115.

Perrin-Jablonski diagram, 516, 517, 697,792.

photobleaching, 697Personal computer (PC), performance

needed for image processing,289–290.

Perspectograph, early studies, 789.Petrán disk, 2, 6, 11, 135, 141, 215,

223–224, 228, 251, 265, 381, 387,447, 458, 554.

Petrán, Mojmir, 2, 6, 11, 215, 223, 228.pH imaging, 188–189, 221, 346, 348, 359,

386, 421, 517, 529–530, 664,739–740, 743, 744.

calibration, 421, 530, 745display, 287intensity image, 529, 530, 739, 740,

744lifetime image, 530

pH indicators, 346, 739–742.pH shift/formaldehyde fixation, 370–371,

373.Phalloidin, as correlative marker, 235–236,

344, 376, 378, 694, 696, 756, 804,854–856.

Pharmacological screening, 813–814.

Phase and intensity determination fromcorrelation and spectrum only(PICASO), 115.

Phase contrast, 9, 171, 368, 372, 453, 506,643, 649, 731, 851, 854, 890, 892.

coherent light for, 130depth of field, 13and holography, 7scanning, 9, 13, 386

Phase fluorometry, 518–519, 526.comparison of FLIM methods, table, 526excitation/emission signals, 519fluorescence lifetime imaging, 518–519

Phase randomization, to scramble light, 8,13, 84, 131–132, 143, 507.

Phase-dependent imaging, depth of field, 13.Phase-shifting interferometry, 245.Phenotyping, 623–624.Phong shading, 308–309.Phong/Blinn models, 308.Phosphoinositide signaling, 799.Photo efficiency. See Photon efficiency.Photoactivatable dyes. See Photoactivation.Photoactivation, 187, 224, 383, 385, 541,

543–545, 693, 759.example, 759genetically encoded

Kaede, 187, 383, 385Kindling, 574, 760PA-GFP, 187, 383, 385, 752, 759–760

table, 385Photobleaching, 174, 218, 224, 275,

341–342, 362–363, 545, 690–700,729, 747–748, 759. See also,Bleaching, and Chapter 39.

autofluorescence, 698defined, 218, 691dynamics, as a source of contrast,

202–203effect on contrast, 174fluorescence intensity loss, 691, 694, 696,

698+

fluorescent image of single protein, 699fluorescent probes, 362–363fluorescent recovery vs. irradiation time,

699fluorophores signal optimization, 341–342

choice of fluorophore, 342fluorophore concentration, 342light collection efficiency, 217, 341protective agents, 36, 341–342, 363,

368, 375, 499, 694spatial resolution, 341

in four-dimensional imaging, 747–748green fluorescent protein (GFP), 690, 692,

698intentional See Fluorescence recovery

afterphotobleaching (FRAP)kinetics, 695mechanisms, 340, 691–693

FRET, 691multi-exponential fluorescent bleaching,

697

multi-photon microscopy (MPM), 545Perrin-Jablonski diagram of bleaching,

697photocycling, fluorescent proteins, 698propidium bound to DNA, plot, 695reactive oxygen species, 341–342,

362–363, 390, 544, 682–684, 691,693–694, 852–853

reduction in, 693–696antifade agents, 36, 341, 368, 375, 499,

694disk-scanning microscopy, 224quantum dots, 694

results, in living embryos, 759of single molecules, 696–698structured-illumination methods, 275two-photon excitation microscopy

(TPEM), 690, 697Photocathode, PMT, 28–29, 232–233.

quantum efficiency, 232–233to reduce transmission losses, 28–29

Photoconductivity, in photodetectors, 252,253.

Photocycling, fluorescent protein molecules,698.

Photodamage. See Phototoxicity.Photodetector. See Detectors; Light

detectors;CCD; EM-CCD; PMT etc.Photodiode, 134–135, 253–255, 610,

707–708.feedback, to stabilize laser, 87, 682feedback, to stabilize arc/filament,

134–135, 137in hybrid PMT, 29, 30infrared sensitive for IR lasers, 707photometer sensor, air space, 26quadrant, for alignment, 87, 134of self-aligning source, 134–135for testing display software response, 830vacuum avalanche, 254, 255

Photoelectric effect, and LED operation,137.

Photoelectrons (PE), 29, 30, 62–63, 77,232–234, 254–255, 257, 259–264,339, 633, 863.

amplification of, 62–63in the CCD, 232–234, 495, 918, 931production in PMT, 30

single-PE pulse-height spectrum, 29, 77secondary electrons, as source of PMTmultiplicative noise, 77

Photoemissive devices, 256–257.Photoemissive effect, 254.Photographic recording systems, 6–7,

11–12, 20, 22, 30, 71–72, 132, 139,141, 162, 207, 217, 263, 280, 488,581+, 588, 590–591, 593–594, 607,613, 628–629, 633, 640, 643, 712,829, 862, 865–867.

“toe” response, quadratic, 71Photometer paddle, to measure light beam,

26, 35, 139–140, 159, 391, 650–651,665.

970 Index

Photometric response, and HD curves, 71.Photomicrography (Loveland), 139.Photomultiplier tube (PMT), 9, 28–31,

35–36, 51, 62–63, 74–75, 222, 232,251, 254, 255, 258–261, 443, 527,661–662.

after pulsing, 257Bio-Rad, 260–261as confocal detectors, pros/cons, 222for epi-fluorescence confocal microscope,

9functioning, 62–63GaAs photocathode, 28–29, 232, 252,

255, 263, 527, 931gain from collisions at first dynode, 31grey levels, 443hybrid, single-pixel signal levels, 31,

254–255linearity, 661–662microchannel plate, 232–233, 255, 262mini-PMT arrays, 51, 667multiplicative noise, 28–30, 77, 633,

677, 926. See also, Multiplicativenoise

in multi-channel detection systems, 51noise and gain, 74–75number of photons striking per unit time,

35–36optical enhancer to increase QE, 28–30photon counting, 21, 29–30, 32–35,

258–259, 260–263, 542quantum efficiency, 527

vs. cooled CCD, 26–28signal variation with time, 232transit time spreads, 527

Photon(s), 20–21, 30, 33–36, 63–64, 132.counting precision, 20–21

uncertainty, 63–64interactions with photomultiplier tube, 30lost, 33–36

Photon counting, 21, 29–30, 32–35,258–259, 260–263, 542.

circuits, 33–34, 258, 521digital representation of optical data,

32–33effects, 34–35examples, 35, 263hybrid PMT, 29–30pile-up losses, 32–33, 35, 78, 521, 523,

526–527with PMT, 29–30, 32–35, 258–259, 260,

263Photon detector types. See Detectors and

entries by each detector type.CCD, 254direct effects, 252image dissector, 254–255microchannel plate, 232–233, 255, 262

MCP-CCD, 262gated, 519, 523–524, 527, 532

photoconductivity effects, 252, 253photoemissive, 254photovoltaic, 252–253

thermal effects, 252vacuum avalanche photodiode, 254, 255work functions, table, 252–253

Photon efficiency, 24–36, 215, 217, 341,631.

defined, 24as a limitation of confocal systems, 24,

223measuring, 26, 33–36, 217practical confocal microscopy, 631of scanners, 215table listing photon losses, 217

Photon flux, statistics, 256–258.Photon interactions, 252–256.Photon (shot) noise, 660–661. See also,

Poisson noise.Photonic crystal fiber (PCF), laser delivery,

1, 88, 109–110, 113, 504, 541.for white light source, 113

Phototoxicity, 112, 363–364, 390–391, 651,729, 746, 770.

chapter, 680–689in brain slices, 729damage is higher to either side of raster,

54factors influencing, table, 363fluorescent probes, 363–364live cells, 390–391reduction, 391from uneven scan speed, 651

Photo-uncaging, 187, 210, 383, 385, 541,544–545, 692, 729, 760, 912. Seealso, Photoactivation.

Photovoltaic effect, 252–253.Phycobiliproteins, 338, 341, 343, 355–357,

693.Physical limitations, 20, 24, 63–64.

on accuracy and completeness of data, 20

Poisson noise, 63–64. See also, Poissonnoise

Physiological fluorescence imaging, 383,385.

PICASO. See Phase and intensitydetermination from correlation andspectrum only.

Piezoelectric effect, defined, 57.Piezoelectric focus controls, 166, 215, 219,

222, 231, 241, 245, 268, 468, 754,909.

Piezoelectric scanning systems, 57, 215,238, 510, 555, 610.

Piezoelectric devices.AOD driver, 54–55, 57acousto-optical components, 54–55, 57to align objective, 166dithering to increase CCD resolution,

70effect described, 57to focus objective, 166, 215, 219, 222,

231, 241, 245, 268, 468, 754, 909laser alignment, 87light scrambler, 84

to move optical fiber, 84to move scanning mirror, 57, 215, 238,

510, 555, 610to move stage, 215, 567phase-shifter

in 4Pi confocal, 609in structured illumination, 268optical coherence tomography,

609–610stretching optical fiber, 609

Pile-up, of pulses.in avalanche photodiode, 253in photomultiplier tube output, 32–35measuring risk of, 34–35

p-i-n diode, 253.Pinhole, 26–28, 33–35, 149, 150, 154, 201,

210, 213, 215, 224–228, 395,631–632.

advantages and disadvantages, 26–28calibrating diameter, 33–34confocal, proper use, 28disk-scanning, 224–228mini-image detection, 32optical fiber as, 506–507optimal size, 226–227, Chapter 22

Fraunhofer formula, 225position in confocal microscope, 210practical, in confocal, 631–632radius, effective, 35ray paths, different sizes, 226–227single-mode polarization preserving fiber,

213small pinholes, effect, 225of tandem scanners, 215vibration shifts relative positions, 201

Pinhole disks, critical parameters, 224–228.Pinhole energy, with spherical aberration,

149, 150, 154, 631–632.penetration into water, 149, 150defocus and NA, 150defocus and wavelength planapochromat,

154Pixel clock, digitization, 62, 64–65, 201,

234–235, 258, 903, 923, 929.CCD, table, 929

Pixels, 38–39, 60, 62–63, 65, 258–259.defining, 60digitization, 62–63optimal, 63–64, 66representing intensity, 258–259and resolution, 38–39and Abbe criterion resolution, 38–39, 65

PKzip, 580.Plan objectives, Zeiss, field diameter, table,

152.Planapochromat, 152, 155. See also,

Objectives.flatness of field and astigmatism, 152lateral chromatic aberration, 155

Plancks law, energy of photon, 35, 137, 252,424.

Planar illumination, SPIM, opticalsectioning, 751.

Index 971

Plane of focus, distortion, 16, 23.by beam deviations, 16by refractile cellular structures, 23

Plant cell imaging, 769–785.autofluorescence, 770–772birefringent structures, 162–164.

420–421. See also, Birefringencechamber slides for plants, 429clearing intact plant material, 166,

417–418, 420, 439, 610, 624,774–775

computer visualization methods, 778deconvolution, 784–785direct imaging, 772–773dynamic imaging, 783–784effect of fixation, 195, 428Equisetum, 774fluorescence properties, 421–428

emission spectra, 421–423microspectroscopy, 421–426

fluorescence resonance energy transfer,425. See also, FRET

harmonic generation See Harmonicsignals fungi, 438–439

genetically encoded probes, 769, 773, 783

green fluorescent protein fusions, 773,783

of green tissues, 770hairs, 434–435history, 769light attenuation in plant tissue, 414–418

A. thaliana, example, 416absorption spectrum, 415effect of fixation, 428maize stem spectra, 417, 418M. quadrifolia spectra, 416M. quadrifolia optical sections, 419Mie scattering, 162–163, 167, 417–418nonlinear absorption, 416–417Rayleigh scattering, 162–163, 167, 417,

703light effects on, 770light-specimen interaction, 425–428living plant cell specimens, 429–439

calcofluor staining procedure, 424, 438

callus, 429cell walls, 168–169, 188–189, 303,

306, 416–417, 420–421, 428–431,435–136, 438, 439, 710–711,713–715, 769–776, 779–781

chamber slides, use, 429cuticle, 434–437, 715, 717, 779fungi, 438–439, 624, 782, 870hairs, 431, 434–436, 772meristem, 168, 420, 430, 770, 776–778,

783microsporogenesis, 431–432mineral deposits, 163, 420, 436–438,

703pollen germination, 420, 433–434, 781,

783

pollen grains, 202, 305, 313, 420,431–433, 553, 558, 781, 783

protoplasts, 195–196, 203, 416, 421,423–427, 429–431, 438–439, 693

root, 172, 174, 303, 307, 421, 429,430+, 438, 464–465, 556, 772–773,775, 777, 779–783

culture chamber, 429starch granules, 202, 420–421, 428,

432–433, 435, 703, 710–712, 715,719

stem, 168, 172, 180, 417–419, 421,424, 429, 556, 707, 710–711,713–714

storage structures, 435–436suspension-cultured cells, 189,

429–430tapetum, 433–434, 779waxes, 420, 428, 434–435, 714–715

new spectral tools, 770obtaining spectral data, methods, 772penetration values, 779photodamage, 770point spread function, 722, 784refractive index heterogeneity, 192,

418–420single-photon confocal excitation,

772–778specific methods, 769spectral unmixing, 770

examples, 665–666staining, 774technological developments, 769textbooks, 769three dimensional, 771

clearing agents, 166, 417–418, 420,439, 610, 624, 774–775

deconvolution protocols, 784reconstruction, 775–776segmentation, 776–778

two-photon excitation, 415–419, 421, 423

advantages, 778–779best conditions, 781compared with one photon, 421cell viability, 779–782deconvolution protocols, 784dyes, 782green fluorescent protein, 782–783light-specimen interaction, 425–427microspectrometer, 424pitfalls, 782thick specimens, 779in vivo, 781

Plasma membrane, microscopy. See Totalinternal reflection microscopy(TIRF).

Plasma light sources, spectra, 44.Plasmid DNA, nick-damage, 684, 724,

802–804. See also, Microinjection;Electroporation; Biolistictransfection.

Plasmodesmata, 777.

Plumbago auriculata, fluorescence spectra,422.

PMT. See Photomultiplier tube.p-n diode, 253. See also, Photodiode.PNG (Portable network graphic), 581, 584.Pockels cell, variable beam attenuator, 25,

54, 57, 87, 116, 543, 701, 903–904.Pockels effect, in crystals, 57.Point-spread function (PSF), 4, 10, 23, 27,

39, 68–70, 145–146, 189–190, 208,223, 239–250, 271, 275, 330, 378,405, 407, 409, 446, 448, 453–457,485–486, 489–494, 536, 562–564,570, 574, 578, 635, 656, 674, 750,784, 830, 895.

3D, 68–70, 247–2484Pi microscopy, 562–563

additional information from, 570space invariance of PSF for, 564

apodization, 240, 243, 249, 250, 272, 567,889

blind deconvolution, 468, 485in botanical specimens, 772, 784in brain slices, 729calculations, RI-mismatch, 407

for glycerol, table, 409for water, table, 409

CARS, 596comparing widefield with confocal, 27,

453–457, 493, 644–647confocal, 10, 12, 208+, 212, 216, 405,

632, 681vs. deconvolution, 27, 453–457, 493,

644–647deconvolution, 189–190, 223, 489,

490–494, 784. See also,Deconvolution

quantifying PSF, 492–494deformation caused by RI anomalies,

22–23Fourier transform, 489, 490lateral resolution. See Lateral resolution

measuring, 240–242, 455, 462, 471,656

amplitude/phase, 242fiber-optic interferometer, 240–241images, 246–248high-NA objectives, 239–250, 492, 656pupil function, 240for 3D deconvolution, 145–146

non-linear, 552, 750and Nyquist, 635, 636, 751, 752optical transfer function, related to,

490–491polarization effects, 249–250pupil function, 245–248. See also, Pupil

functionRayleigh-criterion and Nyquist sampling,

39refractive index mismatch, 405, 407spherically aberrated, 148–150, 407, 492shape in telecentric systems, 208SPIM, 674

972 Index

Point-spread function (PSF) (cont.)STED, diagram, 574, 578structured illumination see Structured

illumination microscopytemperature effects, 25, 85, 248–249, 630terminology, 405Wiener filtering, 494

Points, defined, 59.Point-source, for measuring photon

efficiency, 33.Poisson noise, 20–21, 29, 37, 63–64, 67, 69,

74–75, 81, 164–165, 211, 232, 234,442, 456, 460–463, 468, 487, 495,497, 633–636, 647, 651, 655, 660,693, 784, 835, 923–924, 926. Seealso, Quantum noise, Shot noise.

bleaching, 693of CCD

charge transfer, 920dark charges, 921–922

CT imaging, 615and display linearity, 72–73, 588digitization, as part of signal, 65, 69,

633–636of EM-CCD, 233–235, 262, 927–928and FLIM, 524–525and gray levels, 74importance of deconvolution, 38–41, 60,

69, 189–190, 222–223, 320, 399,471–472, 481, 495, 751–753, 835

intensity spread function, 75–78photomultiplier tube, 74–75

affects effective QE, 31multiplicative noise, 29, 647, 660

in photon detection, 63–64and pixel size, 64, 68, 633–636, 928practical effects, 67single-pixel noise, 65, 67, 190, 635, 832,

835–836spectral unmixing, 667, 770

examples, 665–666structured illumination, 278uncertainty in contrast, 74, 164–165and visibility, 37, 667

Polarization, 13, 49, 57, 83, 88, 89,211–212.

attenuator, 43, 543, 907beam-splitter, 13, 50–51, 57, 85, 87, 100,

171, 217, 513, 631, 904to avoid spectral distortion, 49

circular or phase randomized, 211–212,229

effect on AODs, 55effect, of dichroic beam-splitters, 34,

49–50Kerr cell, 111, 113, 516of laser light, 8, 83, 88–89, 113, 478,

558optical components, 57, 155, 211optical fibers, 213Pockels cell, 25, 54, 57, 87, 116, 543,

701, 903+

rectified DIC optics, 846

to reduce reflections, 6, 25, 141, 158, 171,516, 229. See also, Antiflex system

scramblers, 8, 84, 132, 143Polarization effects, 211, 249–250, 503.

birefringence, 188, 420–421, 431, 434,436, 438, 480, 503. See also,Birefringence

blind deconvolution, 479and CARS microscopy, 595, 600–604high-NA objective lenses, 249–250,

267interaction with nucleus, 23optical fibers, 503, 507stereo image displays, 299, 589

Polarization microscopy, 43, 50–51, 154,156, 162, 188, 288, 348, 438,479–480, 513, 555, 711, 714–715,717, 719, 891, 894.

centrifuge microscope, 8of collagen fibers, 164, 188, 717DIC, 10, 14, 127, 146, 468, 473and FRET, 793and harmonic generation, 179, 428,

704–706, 717, 719MFMP, 555mitotic apparatus, 15, 717p- and s-, and incidence angle, 50–51Pol-scope, 13, 188, 432, 468, 480PSF, 406–407to regulate light intensity, 43STED, 578

Polarization noise, in lasers, 83.Polarization-preserving fiber, 49, 87, 503,

505, 507.as a pinhole, 213

Polarization-sensitive detection CARS (P-CARS), 600, 601, 604.

adipocyte cells, 604Polarized light, 7, 14, 83–85, 146, 158, 162,

171, 229, 406–407, 420, 479, 894.deconvolution, 479image formation, 406–407PSF, 479

Polarizer, 83, 128, 188, 249, 268, 275, 420,479, 711, 903–904.

for antiflex, 6, 84, 141, 158, 229for attenuation, 43, 85, 87–88, 543,

903–904for CARS, 601Glan-Taylor, 85, 87, 100, 171Glan-Thompson, 85, 904LCD, 589, 715micro-wire, 85structured illumination, 264tunable, 715

Pollen germination, 433–434.Pollen grains, 202, 305, 431–433, 438, 553,

556, 558, 678, 781, 783.germination, 433–435, 783–784multi-focal multi-photon imaging, 556Pol-scope, 13, 188, 432, 468, 479–480test specimen, 195, 269, 313, 553, 556,

678

Pol-scope, 13, 188, 432, 468, 479–480.deconvolved, 479images, 15, 188, 479, 717

Portable network graphic. See PNG.Position, accuracy in CLSM, 40.Position sensors, galvanometer, 53–54.Posterizing, 591.Potassium titanium oxide phosphate (KTP)

crystalfor non-linear optical frequency conversion,

107.Potato (Solanum tuberosum) SHG signal,

712.Power requirements, for lasers, 65, 80–81.Power spectrum. See Contrast transfer

function.Power supply, laser as noise source, 86.PP, Periodically poled waveguide,

114–115.Practical confocal, 2-photon microscopy,

tutorial. See also, each topic as amajor entry.

2-photonexcitation duty cycle, 644peak power level, 644photodamage vs. penetration, 645power vs. penetration, 646

3D microscopy methods compared, table,647+

best 3D method for, 644–647biological reliability, 631bleaching pattern, 627–628

quantum efficiency, 628chapter, 627–649confocal images with few photons, 634deconvolution, factors, 646filling back-focal plane, 210, 509, 629,

633focus, compensating drift, 395, 732getting a good confocal image, 629–631

alignment of optics, 629–630back-focal plane (BFP), 210, 509, 629,

633focus, 629low signal, 631mirror test specimen, 630no signal, 631, 660simultaneous BSL/fluorescence,

631getting started, 627Köhler illumination for transmission, 34,

127–128, 131, 229, 627, 648–649multi-photon vs. single-photon, 646new controls, 631–636

biological reliability, 631pinhole size, 631–632pixel size, 62, 634–635, 784, 928

Nyquist reconstruction/deconvolution,635–636

over-sampling, 635photon efficiency, 24–26, 215, 217, 341,

631pinhole summary for, 26–28, 633

Index 973

pixel size, 62, 634–635, 784, 928measuring, 635summary for, 636

poor performance, reasons, 640–643air bubbles, 643curvature of field, 641dirty objective, 642–643imaging depth, 643under filling objective pupil, 642optical problems, 640–641sampling problems, 640singlet-state saturation, 643

under-sampling, 635schematic diagram, 632statistical considerations, 633–634stray light, 201, 632, 904test specimen, 636–640

description, 636–637diatom, 638–640figures, 637–640reasons for, 636

widefield vs. beam scanning, 647Prairie Technologies, LiveScan Swept Field

design, 237.Pre-amplifier, in digitizing analog signal,

64.Precompensation, in fiber optic cables, 88.Presentation software, 829–845.

helpful URLS, 844–845movies, 837–844

artifacts, 839–840coding limitations, 838compression of large movies, graph,

843compression of PAL TV movies, table,

842digital rights management, 844entropy, 841frame count matching display cycle,

838–839MPEG display formats, 840–841overlaying, 844Pack-and-go mode, 842, 844performance benchmarks, 841–842region code, 844remote use, 842–844rules 837–838, 844up-sampling, 838–839very high resolutions, 841

precautions, 829–830testing, 830–836

aliasing gallery, 834aligning images, 835brightness, 832changing display size, 832–835codecs, 831compression, 835–836compression artifacts, 837cropping, 835digital rights management (DRM), 830down-sampling in PowerPoint, 834fast graphics cards, 831, 832gamma, 832–833

measuring display speed/sensitivity,830

random color dot image, 836reference images, 830–831removing distortion, 835resolution, 832–835rotating, 835scaling, 835screen capture, 830static image performance, 831step image, 833under-sampled image, 835up-sampling, example, 834viewer, 830

Preventive maintenance, lasers, 115–116.Principal component analysis (PCA),

731–732.Printers, 591–593.

aliasing, 592color images, 592grey levels, 592ink jet, 593laser, 593posterizing, 591scaling techniques for, 592

Prionium, MMM image, 556.Probe, mismatch with pixel shape, 39.Processor performance, 3D-image display,

289+.Projection/compositing rules, 3D-image

display, 302–304, 763–764.alpha blend, 302, 304average intensity, 302first or front intensity, 302Kalman average, 304maximum intensity, 302

Propidium iodide, 344, 355, 360, 426, 651, 693–695, 773, 778–779, 782,812.

dead cell indicator, 426, 651, 875, 877Proteins, 195, 756, 760, 794–795, 804. See

also, Green fluorescent protein, etc.chimeric fusion, 794fluorescent, FRET, 794–795Kaede, 187, 383, 385Kindling, 760microinjection, 804PA-GFP, 187, 383, 385, 752, 759–760tagged, 756, 758translational fusions, 756UV absorption, 195

Proteomics, 237, 790, 804, 809, 818, 867.location, 825

Protoplasts, 195, 416, 429, 430, 431.A. thaliana, 195–196, 203, 416, 421,

423–427, 429–431, 438–439, 693Proximal tubule, labeled, 744.Pseudo color display, 173–175, 190, 291.PSF. See Point spread function.Pulse broadening, 88, 111, 210, 537–538,

543, 606, 609, 728, 903.Pulse length measurement, 115, 901–903.Pulse spreading. See Pulse broadening.

Pulsed lasers, 81, 96–100, 110–114, 120,137. See also, Lasers; Ultrafastlasers.

broadband tunable, table, 120diode, table, 96–97DPSS, table, 98dye, table, 96excimer, table, 96for FLIM, 537kits, table, 98, 100nitrogen, table, 96scanning only region of interest, 237for 2-photon excitation, 81ultrafast, table, 99–100vapor, table, 97

Pulse-counting mode, 21, 29–30, 32–35,258–259, 260, 263.

Pump sources, for dye lasers, 103.Pumping media, maintenance, 116.Pumping power vs. frequency cubed, 65, 82.Pupil function, 211, 245–248.

3D point spread function restored,247–248

4Pi, 566–567AOD, 56empty aperture, 248of human eye, 72, 128intermediate optics, 211, 222, 225, 250Köhler illumination, 34, 127–128, 131,

229, 251, 627, 648–649Mach-Zehnder interferometry, 245measurement, 246–248

images, 246–248objective, 24, 155, 158–159, 211,

239–240, 242, 492, 551–552, 554,566–567, 650

orthonormal Zernike polynomial for,table, 247

phase-shifting interferometry measuring,245

polarizing effects, 249pupil plane, 50 See also, Back-focal plane

transfer lens, 728view of pupil image, 629Zernike polynomial fit, 245–247

Purkinje cells, Golgi-stained, 167–168.Pyrus serotina. See Pear.

QQE. See Quantum efficiency.Q-switched pulsed laser systems, 111,

114–115.Quantitative analysis, flying-spot

microscope, 6–7.Quantization, limitations imposed by, 37–39.

See also, Chapter 4.Quantum dots, 221, 343, 357–358, 360–361,

656, 694, 696, 757, 801, 814, 846,853. See also, Semiconductornanocrystals.

assays for, 814in electron microscope, 852–854FRET, 801

974 Index

Quantum dots (cont.)labeling, 853toxicity, 357, 694

Quantum efficiency (QE), 25–30, 74–78,222, 232–234, 238, 251, 254–255,349, 355, 375, 383, 390, 442–443,459, 516, 527, 575, 628, 646, 556,703, 751, 793, 920–922.

of back-illuminated CCD, 77–78charge-coupled device (CCD), 26–28,

74–76, 142, 215, 232, 234, 257–258,261, 644, 707, 751, 754, 810,920–921

comparative among CCD cameras, 76effect on Poisson noise, 74–75effective QE, of photon detectors, 28, 29of electron-multiplier CCD, 4, 30, 59,

234, 920FLIM, 516–517, 520, 523, 526–527, 529,

530FRET, 792of human eye, 251and intensity spread function, 74–75and multiplicative noise, 77optical enhancer, to increase QE, 28–30optimal 3D microscopy, 644photomultiplier tube (PMT), 26–28, 51,

77, 222, 257, 262, 527, 707graph, 29table, 707

signal-to-noise ratio, 263, 442–443variation with wavelength, 29vs. wavelength, 922

Quantum noise, 21 63–64, 69–70, 468, 472.See also, Poisson noise.

and approximation, for reconstruction,69–70

Quantum wells, as absorbers, 111.Quantum yield, of fluorescent dyes, 172,

180, 338–345, 353, 360, 363, 383,421, 543–544, 574, 661, 683, 690,710, 737, 792, 794–795.

Quartz-halogen lamp, control, 138–139. Seealso, Halogen lamps.

RRabbit, 237, 744.

antibodies, 855, 877–878kidney proximal tubule, pH, 744

Radiance, of non-laser light sources, 126,132, 137–139, 141.

measuring with radiospectrometer, 139

Table, 1140Radiospectrometer, radiance vs. wavelength,

139.Raman background, in glass fibers, 88, 90,

162, 506–507.lower in large-mode-area, fiber, 110

Raman scattering, 162–163, 167, 339–340,348, 506–507, 545, 697.

and bleaching, 697defined, 162

Raman spectroscopy, 48–49, 90, 167, 254,339–340, 507, 545, 697. See also,CARS.

CARS, 204, 550, 577, 595–605chemical imaging, 90hard-coating on interference filters used,

48–49image contrast, 167

Ramp-up, for light sources, 136, 137.and long-term stability, 137and short-time stability, 136

Rare earths, for doping fiber lasers, 110.Raster, 62–64.

convolution, 485–486dimensions, in specimen, 63retrace, 25, 33, 53–54, 219, 338, 389,

543, 628, 651, 908. See also,Retrace, raster scanning shape, 63

size, vs. pixel size and light dose, 64temporal limitations, 141

Raster scanning, 5–6, 25, 141–142, 223,540, 596.

alignment, 629–630, 651assymmetrical sampling, 38–40bleach pattern, 3D, 538, 628, 693chromatic aberration limitations, 156,

640–641damage is higher to either side of raster,

54display, 830–831, 835distortion, 40and electronic bandwidth, 70, 238for fast confocal imaging, 223fiber-scanning, 214, 508galvanometer limits, 52–54, 223, 651limitations imposed by AODs, 56MPEG formats, 840Nyquist sampling, 38, 41, 59–60, 62,

634–635off-axis aberrations, 151, 640–641,

659–662pattern on Nipkow disk, 5–6, 223–225.

See also, Nipkow disk scanningretrace gating, 25, 54, 56, 219, 389, 543,

628, 651, 908scan angles, 209, 214stability, 708sampling in time and space, 141–142timing, 33, 53, 753zoom, raster size and magnification, 11,

24, 37, 63–64, 66, 70, 79, 317, 389,493, 627, 634–636, 655–658, 683,731

Rat, cells and tissues, 205, 320, 323, 330,398, 739, 813.

brain slices, 393, 398, 686CA1 region, 323

cardiac muscle, 498, 529, 556cerebellar granule neurons, 813EDL muscle, calcium, 740fixation, 370, 372, 393hippocampus, 268, 317, 341

interossi muscles, SNARF-1, pH image,739

intervertebral disk, 310–311kidney, 511, 803leukemia cells, 347, 520–521

FLIM image, 521neuron, membrane potential, 205tooth, 667

Rate, imaging, limited by signal level, 73.Ratiometric imaging, 189, 346–347. See

also, Calcium imaging, pH, etc.bleach ratio, 697–698calcium, 736–737, 850. See also, Calcium

imagingCARS, 600, 602, 604. See also, CARSconcentration calibration, 742–745to detect colloidal gold labels, 167to determine ionic concentration, 36FLIM, 516–532. See also, FLIMFRET, 174, 184, 790, 794–795, 797–798.

See also, FRETglutaraldehyde autofluorescence assay,

369HCS, high-content screening, 813,

823–824.indicator choice, 738interpretation, 740–741live/dead assay, 875pH, 739–744. See also, pH imagingstructured illumination. See Structured

illumination microscopywater-immersion objectives, 737

Rayleigh criterion (Abbe criterion), 1–3, 9,37–39, 60–61, 66, 129, 146, 486,703, 822, 928.

breaking the Abbe/Rayleigh barrier,571–573

Nyquist sampling, 39, 60, 66of two point images, 1–3, 146

Rayleigh scattering, 162–163, 166, 167,339, 342, 417, 703, 747.

compared to Mie scattering, 163in embryos, 747by colloidal gold labels, 167light attenuation in plant tissue, 417wavelength dependency, 162–163

Rayleigh unit, 147.Reactive oxygen species (ROS), 341–343,

362–363, 390, 544, 682–684, 691,693–694, 852–853. See also,Bleaching; Phototoxicity.

as basis of correlative TEM staining,852–853

Readout noise, 74–75, 77, 232. See also,Noise.

and readout speed, 77Real image, disk-scanners, 224.Real-time 2D imaging, 12–13, 167–168,

215, 222–224, 232, 235, 307, 496,542.

Real-time 3D imaging, 154.Receptors.

cholera toxin, 790–791, 796–797, 802

Index 975

deconvolution, 495EGF, 533ERD2, 791, 796fibrinogen, 846–847, 850high-content screening, 809, 812–814KDEL, 790, 797ligands, 354lipid, 790, 791proteins, 357Streptococcus, 879transferin, 819uncaging, 545

Reconstruction, 3D.definition, 280.Nyquist and filtering/deconvolution, 59,

66–67, 69, 70, 173, 235–236,280–315, 458, 468–469, 474–475,496–497, 563, 585, 603, 607, 610,615, 635, 672, 675, 677–678, 690,772, 730, 731, 762, 77, 774–776,778, 784, 883

Recording times, 141–142.in widefield microscopy, 141–142using LED source systems, 141–142

Recovery curve, after bleaching, 187.Red fluorescent protein (RFP), 221–222.Reference list.

historic, 889–899lasers, 123–125

Reflected-light images, 180, 181. See also,Backscattered light.

confocal, of integrated circuit, 180of glass bead, in water, 181

Reflecting objectives, constraints, 156.Reflection contrast technique, Antiflex, 159.Reflection mode, low coherence light, 130.Reflectivity, optical surfaces, 159, 163,

167–171.anti-reflection coatings, 158

on-axis, artifact, 168–171refractive index, 159, 163, 167

Refracting regions affect imaging beam,15–16.

Refractive index, (RI), 14–15, 23, 45, 148,152, 163, 198, 377, 404–413,418–420, 613, 654. See also,Spherical aberration; Dispersion.

anomalies in, effect on PSF, 23, 418–420of biological structures, 163, 377

table, 277of botanical specimens, 418–420coverslip thickness, importance, table,

654of immersion medium, 277, 411

effect on PDF, 23, 418–420effect on sharpness, 14–15effect of wavelength and temperature

on, 148, 248–249, 411and intensity, and spectral broadening,

111of layers in interference filters, 45of mounting media, table, 198, 342,

370–371, 373–377

of optical glass vs. wavelength, 152optical projection tomography (OPT), 613self-shadowing, 198temperature, 148, 248–249, 411of tissue/organs, table, 377

Refractive index mismatch, effects,404–413. See also, Sphericalaberration.

table for glycerol, 409table for water, 409calculation, 404–407dependence of focal shift, 410diagram, 404dry objectives, 410–411experiments, 409–410

water/glycerol results, table, 410field strength calculation, 405other considerations, 410–413spherical aberration correctors, 15, 151,

147, 192, 411–412terminology, 405

actual focal position (AFP), 405focal shift, 405nominal focal position (NFP), 405

theory, 404–407Region code, for MPEG-encoded movies,

844.Region-of-interest (ROI), 835.

brain slice, 726, 733diagonal, 658display presentation, 835embryos, 747, 759FRAP, 51, 187FRET, 797, 801in image processing, 289, 300, 323, 330,

676labeling, 353must be smaller at high resolution, 577nanosurgery, 219, 686photobleaching, 690preprocessing, 676rapid acquisition, 236–237structured illumination, example, 272viability studies, 683

Registration synthesis method, 328–331.defined, 328landmark-based, 328–329multi-view deconvolution, 330

Relationships, in fluorescence microscopy,80.

energy per photon, 80flux per pixel, 80photons/s vs. wavelength, 80

Relative motion, objective vs. specimen,39–40.

Relaxation, in laser energetics, 82.Relay optics (telan lenses), 145, 157, 214,

455.Reliability.

of 3D image, 461, 517biological, vs. damage, 24, 68, 631, 633lasers, 80, 102, 115living cell work, 387

mirror position, 40photometric, 312spectral detectors, 662

Removable storage media, 585–588.random-access devices, 586–588

compact disks (CD), 586–587digital video disks (DVD), 587–588floppy disks, 586magnetic disks, 586MO (magneto-optical) disks, 586optical disks, 586WORM (write once, read many) disks,

586Rendering, of 3D views, 280, 285, 290, 301,

307, 309, 311, 377, 749, 762, 764.definition, 280voxel speed, 290

RESOLFT microscopy, 571–574, 577. Seealso, STED.

breaking diffraction barrier, 571–573concept, 571–573different approaches, 573–574

ground state depletion (GSD), 573STED, 573–574

outlook for, 577resolution, new limiting equation, 571triplet-state saturation, 573

Resolution, 1, 4, 13, 16, 24, 36–41, 59, 61,65–67, 210. See also, PSF; FWHM.

adequate levels, 36–41axial, 13axial-to-lateral ratio vs. NA, 4back-focal plane diameter, table, 210confocal vs. non-confocal, 16and contrast transfer function, 37, 59, 61estimating, 65–67measured, widefield, 16minimum resolvable lateral spacing, 1, 16spatial and temporal, 24sufficient, 36–37

Resolution, structured illumination.Fourier-space, 270–271linear image reconstruction, 271Lucosz’s formulation, 273methods, 270–276Moiré effects, 270–271photobleaching, 275reconstruction results, 272standing-wavefield microscope, 275thick samples, 274, 275, 278–279

Resolution scaling, STED comparison, 578.Resolution test slides, 16, 656.Resonant cavity, laser, 81–82, 111, 115.Resonant scanners, 52–54, 56–57, 223, 447,

543.acceleration distorts mirror shape, 53blanking, 25, 218, 338, 389, 543, 628,

651, 908compared to acousto-optical deflector, 56duty cycle, 52galvanometer, 52

multi-photon excitation, 543raster-scanning, 33, 53–54, 56

976 Index

Resonant scanners (cont.)retrace, 54, 56. See also, Retrace, below.scan speed, 54

Retrace, raster scanning shape, 25, 54, 56,219, 389, 543, 628, 651, 908.

acousto-optical deflector, 56blanking, 25, 219, 338, 389, 543, 628,

651, 908raster-scanning, 33, 53–54, 56

Review articles, listing, 889.RFP. See Red fluorescent protein.Rhodamine, dyes, 81, 109, 116, 136, 140,

203, 264, 292, 339, 342–345, 353,355, 362–363, 375–378, 409, 538,553, 592, 693, 697–698, 762,783–784, 794, 851, 854–856.

arsenical derivatives, 348bleaching, 697, 698calibration plot, 661, 851excitation of, 181, 109fluorescence correlation spectroscopy,

693FRET, 347photobleaching quantum yield, 363planar test specimen, 538power for 1-, 2-photon excitation, 81, 3

41Rhodamine-123, 374, 389resolution measurement, 409stability and cost, 116

Rice (Oryza sativa), 168, 171, 414, 415,712, 715.

absorption spectrum, 415, 706backscattered light image, 168, 171emissions spectra, autofluorescence, 713leaf fluorescence images, 714–715light attenuation in plant tissue, 414silica deposits, 714–715, 717

Richardson-Lucy, deconvolution, 497, 568.Richardson Test Slide Gen III, 652, 656.RLE. See Run-length encoding.RNA, microinjection of, 803, 804.RNA labels, 344, 369, 465, 531–532, 612,

691, 758, 874–875.ROI. See Region of interest.Room light, as stray signal, 201, 632, 904.Roots, plants, 172, 174, 303, 307, 421,

430–432, 438, 464–465, 556,772–773, 775, 777, 779–783.

maize, image, 432mounting, 429, 431

ROS. See Reactive oxygen species.Rose Criterion, 37–38, 68, 164, 633.

relationship with signal-to-noise ratio,164

for visibility, 37Rotating, specimen, 188, 568, 835.

micro-CT, 615optical projection tomography, 610–611SPIM, 672–673, 676, 751

Rotor, galvanometer, detecting position,53–54.

Run-length encoding (RLE), 580.

SSafety, 83, 85, 90, 115, 117–118, 124,

132–139, 900, 903, 904.arc sources, 132–139beam-stop design and use, 118. 903–904classification of laser systems by hazard,

117cleaning objectives, 642display geometry, 297equipment needed, 900eye protection

against Brewster surface reflections, 83goggles, 118with external-beam prism method, 90

fiber optics for transporting laser light, 88

hazardous materialsfluorescent laser dyes, 85, 103, 116used beryllium oxide tubes, 115

high pressure Xe lamps, 136monitor power to avoid explosions,

138–139in disk-scanning confocal microscope,

231laser, 117–118, 839, 900, 903–904

installation requirements, 85monitor power to avoid explosions,

138–139references, list, 123safety curtains, 117, 904training, 118

SAM, saturable absorber mirror, 111.Sampling. See Digitization, 20, 63–64.

non-periodic data, 38optimal, 63

Saponin, formaldehyde fixation, 359, 375,856.

Saturable absorber mirror, pulsed lasers,111.

Saturable Bragg reflector (SBR), 111.Saturable output coupler (SOC), 107, 111.Saturation, singlet-state fluorescence, 21–22,

41, 142, 265, 276, 339, 442, 448,450, 643, 647, 899.

performance limitations, 81, 450, 928SBR, saturable Bragg reflector, 111.SBT. See Spectral bleedthrough.Scaling techniques, 592, 835.Scan angle, and position in image plane,

209–210.Scan instability, detecting, 40–41.Scan raster, testing, 651–654.

malfunctioning system, 653phototoxicity from uneven scan speed,

651sources of fluorescent beads, table, 653well-calibrated system, 652–653x and y galvanometers, 651–652z-positioning calibration, 652, 654

stability, 652Scanned-slit microscopes, table, 224.Scanner arrangements, evaluation,

213–215.

Scanners, 51–55, 57, 214–216.acousto-optical deflectors, 55. See also,

AODsmirror arrangements, 214evaluating, 215–216mechanical, 51–54. See also,

Galvanometerspiezo-electric, 57, 215, 238, 510, 555,

610single mirror/double tilt, 215sinusoidal, “tornado” mode, SIM scanner,

52Scanning electron micrographs, 428, 434,

437, 846–848, 850–852.Scanning laser ophthalmoscope (SLO), 480.Scanning fiber-optical microscopy. See

Fiberoptic confocal microscope.Scatter labeling for tracing lineage, 461,

462.Scanning systems for confocal light

microscopes. See also,Galvanometers; Disk-scanningconfocal microscopy; Acousto-optical deflectors; Linescanningconfocal microscopes; Raster.

Lissajous pattern, circular scanning. 554“tornado” mode, SIM scanner, 52

Scattering, 162–163, 167–171, 550.coherent anti-Stokes Raman (CARS), 550elastic, Rayleigh, 162–163, 166–167, 339,

342, 417, 703–747Raman, 162, 167, 339–340, 348,

506–507, 545 and reflection contrast,167–171

Scattering object, viewed by TIRM 177. Seealso, Backscattered light.

Schiff reagents, 262, 369, 770, 774–775,778.

Schottky diode, photodetector, 253.Scientific thought, four aspects, 789–790.Scion Image, 281–282, 395, 730.Scramblers, light, 8, 13, 84, 131–132, 143,

507.Screen capture, 830.Screens, to enclose laser beams, 118.SD. See Static discharges.SDA. See Stepwise discriminant analysis.Sea urchin, S. purpuratus, 173, 198, 200.Second harmonic generation (SHG), 90,

114–115, 166–167, 179, 188, 550,552, 556, 703–719, 729–730. Seealso, Harmonic signals.

as autofluorescence, 361cell chambers, 166, 429, 552detectors, 706–708, 728

disk-scanning, 552, 556double-pass detection, 166–167table, 706–708

crystals for SHG, 103, 107, 114–115, 188,703

energy relations, 705in lasers, 103, 107, 114–115layout, 166, 191, 552, 708–709, 712

Index 977

light attenuation spectra, 706light sources, 706–708

brain slices, 729–730non-linear optical microscopy, 704–705optically active animal structures in,

714–717brain slice, 729–730collagen structure, 703, 717sarcomeres, 716spindle in mouse zygote, 717spindle in zebrafish embryo, 718structures producing SHG, table, 715table of structures, 715zebrafish embryo, 716, 718

optically active plant structures, 428,710–714

Canna, nonlinear absorption, 710cell wall, 428, 711, 714Commelina communis, 712emission spectrum of maize, 710, 711Euphorbia pulcherrima, spectrum, 710mineral deposits, 436Pyrus serotina, spectrum, 711rice leaf, 712, 715starch granules, 433

maize, 710–711, 713–714emission spectrum, 710–711leaf spectrum, 710pol-microscopy, 711stem, optical section, 714stem, spectrum, 710, 713chloroplasts, tumbling, 713

membranes of living cells, 90mineral, deposits, 436photodetector suitability, table, 706–707polarization dependence, 71, 717–720potato, as SHG detector, 712pulsed laser suitablity, table, 706signal generation, 179, 552, 597, 704–705spectra, 706spectral discrimination, 421starch granules, 433

Segmentation, FLIM image, 527–528.Segmentation methods, 281, 283–285, 290,

300–302, 304–306, 309, 311–312,316–319, 321–330, 333–334,527–528, 776–778, 812.

3D, 776, 822, 828automated, 818, 821–822, 828background, 321blob segmentation example, 322–324

gradient-weighted distance transform,323

model-based object merging, 323–325watershed algorithm, 323–324

bottom-up, 321combined blob/tube segmentation,

328–330foreground, 321hybrid bottom-up/top-down, 322integrated, 322intensity threshold-based, 321object, 321

for plant cells, 774–777balloon model, 776watershed algorithm, 322–325, 777,

822region-based, 321–322top-down, 322tube-like object segmentation, 324–328

mean/median template response, 328skeletonization methods, 324–325vectorization methods, 324–327

validation/correction, 333–334manual editing, 333–334

Selective plane illumination microscopy(SPIM), 613, 614, 672–679, 751.

3D scanning light macrography, 672anisotropic resolution, 678applications, 675axial resolution, 674–675vs. CLSM, 678Drosophila embryogenesis, 675–676,

747–748, 751–752, 754, 756, 759,804, 810

and FLIM, 527images processing, 675–678

image fusion, 676–677pre-processing, 676registration, 676

lateral resolution, 674light-sheet illumination, 672–674light sheet thickness, 674–675Medaka, 614–615

heart image, 614embryo image, 675

multi-view reconstruction, 675–678point spread functions (PSF), 674schematic setup, 613, 673thin, laser light-sheet microscope,TLLSM,

672Self-aligning arc source, 135.Self-shadowing, 165, 174, 194, 195.

in confocal optical sections, 174spherical structure, 195

in epi-fluorescent mode, 165, 194SEM. See Scanning electron microscope.Semi-apochromat, pros and cons, 158.Semiconductor lasers, 86, 105–108.

noise sources, 86Semiconductor nanocrystals (quantum dots),

221, 343, 357–358, 360–361, 656,694, 696, 757, 759, 801, 814, 846,853.

as probes, 221, 757, 759Semiconductor saturable absorber mirror

(SESAM), 107, 111.for self-starting intense optical pulse

trains, 111Sensitivity, video photodetectors, 6–7.Sensitized emissions, of acceptor, 795–796.

See also, FRET.Sequential devices, 585–586.Serial sampling, single-beam confocal, 20.SESAM, Semiconductor saturable absorber

mirror, 107, 111.

SFP. See Simulated fluorescence process.Shannon, Claude, 64–65.Shannon sampling frequency, defined, 64,

443.SHG. See Second harmonic generation.Shift invariance, deconvolution, 457, 490,

564.Short-pass filters, 43–44.Shot noise, 232, 256–257, 286, 442,

460–461, 495, 558, 660–661, 831.See also, Poisson noise, Quantumnoise.

Signal, 27, 62. See also Speed relationshipto magnification, 62

Signal attenuation-correction, 320–321.Signal detection, basics, 660–663, 918–931.

See also, Detectors.coefficient of variation, 660instrument dark noise, 660photon (shot) noise, 660–661PMT linearity, 661–662signal-to-noise ratio, 660spectral accuracy, 662spectral resolution, 662–663wavelength response, 663

Signal levels, 16-photon peak signal, 73–74.Signal-to-background ratio, of titanium-

sapphire laser, 112.Signal-to-noise (S/N) ratio, 37, 53–54, 67,

81, 164, 251, 257, 265, 330, 340,386, 391, 442–451, 470, 481, 495,498–499, 528, 542, 562, 567, 582,599, 621–622, 660, 690, 696, 699,707, 736–737, 740, 753, 769, 772,778–780, 810, 813.

3D imaging, 448–4514Pi microscopy, 562–567bleaching, 391, 442, 690, 696in calcium imaging, 737chapter, 442–451comparative performance, 256, 448–451

bleaching-limited performance,448–450

configurations of microscope, 448, 449disk-scanning microscope, 449line illumination microscope, 449saturation-limited performance, 450scanning speed effects, 53, 450–451structured illumination, 265–266, 270,

275–276, 279–280S/N ratios for, table, 450widefield (WF) microscope for, 450

confocal microscope, 444–447, 660calculations, 444detectability, 446–447methods compared, 450noise model N1, 444–445noise model N2, 446–447

deconvolution, 470, 481, 495, 498–499designs, confocal, 212–216, 447–448, 450disk-scanners, 221dynamic range, 2-photon, 644, 778–780high-content screening, 810

978 Index

Signal-to-noise (S/N), ratio (cont.)improvements, 736micro-CT, 615magnetic resonance microscopy, 621–622multi-photon fluorescence microscope,

112, 427, 447, 542, 779Nyquist sampling, 67, 448optimal excitation power, 81, 340Rose criterion, visibility, 37–38, 68, 164,

633saturation, 442vs. scan rate, 53signal level, 67, 75, 528sources of noise

background noise, 443–444grey levels, 443quantum efficiency, 442–443shot noise, 442–443

sources of noise, 442–444STED, 574and visibility, 37

Silica glass, transmission losses, 502.Silicon diodes, near infrared emission, 132.Silicon-intensified target (SIT) camera, 730.

brain slices, 730SIM. See Surface imaging microscopy.Simplicity, as design goal, 43, 66, 220, 229,

387, 508, 647.Simulated fluorescence process (SFP), 310.Single-cell automatic imaging, 809, 812.Single-cell calcium imaging, 812.Single-longitudinal-mode fiber laser, 110.Single-mirror/double tilt scanner, 215.Single-molecule, 80.

biochemistry, 221–222, 575, 690, 693,696

bleaching, 690, 693, 696, 697–698, 699Single-photoelectron pulse heights, 30.Single-photon, energy, equation, 35.Single-photon counting avalanche

photodiodes (SPAD), 527.Single-photon excitation, plant imaging,

772–778.Single-photon pulses. See Photon counting.Single-scan images measure scan stability,

40–41.Single-sided disk scanning, confocal

microscopy, 132, 141–142, 168, 171,175, 215–216, 229, 231, 907, 913.See also, Disk-scanning confocalmicroscopy.

advantages and disadvantages, 215–216basic description, 141commercial, 907, 913light source, 132, 141–142

Singlet state saturation, 21–22, 41, 81, 142,265, 276, 338–339, 442, 448, 450,643, 647, 899, 928.

Sinusoidal bidirectional scanning, 25,52–54. See also, Resonant scanners.

duty cycle, 53, 260Sinusoidal image, 831, 838.

fiber-optic confocal, 510

Sinusoidal modulation, in FLIM, 524–526.SIT. See Silicon-intensified target camera

imaging.SLF. See Subcellular location features.Slice AM-dye-painting protocol, 726–727.Slice chamber protocol, 727.Slit scanning confocal, 12, 25, 37, 50, 51,

56, 221–226, 231+, 235, 238, 519,522, 664, 741, 914, 916.

Achrogate, 50, 212, 231–232, 916with AOD scanning, 56, 914commercial, 913–914, 916critical parameters, 224–228optical sectioning, 228, 444–449optimal slit size, 225point excitation, slit detection, 914

SLM. See Spatial light modulator.SLT. See Subcellular location tree.Smart media, digital storage, 588.SMD. See Surface mount device.SNARF-1, 345, 346, 531, 739, 744–745.

ratiometric pH label, 744–745stained rat interossi muscles, 739table of variants, 531

Snell’s law of refraction, 167, 654.SOC. See Saturable output coupler.Software packages, visualization, table,

282–283.SoftWorx, 3D display software, 282.Solanum tuberosum, potato, 712.Solid state memory devices, 588.

compact flash cards, 588memory stick, 588smart media, 588

Solid-state photodetector, 30–31, 918–931.See also, CCD; EM-CCD.

Solid-state lasers, 86, 103–118, 236–237.cooling, 108noise sources, 86thin-disk lasers, 109tunability, 109use, 236–237

Source brightness, measure, radiance units,126.

Source optics, reflecting and collecting light,134.

Space invariance, telecentric systems,207–208.

Space multiplexing, in MMM, 555.Spacer, material in interference filters, 46.SPAD, single-photon counting APD, 527.Spatial coherence, 84.Spatial filter, 89, 107, 391, 542, 708, 729.

optical devices for, 89, 222–223, 729digital, 391–392. See also, Gaussian

filteringSpatial frequency, 37, 60, 65, 66. See also,

CTF.and contrast transfer function, 37and geometry, 66response of microscope, and pixel size,

65zero, as measure of brightness, 60

Spatial laser beam, characteristics, 89.Spatial light modulator (SLM), 266.Spatial orientation factor, for FRET,

792–793.Spatial resolution, in confocal microscopy,

24. See also, Resolution, PSF, CTF.Special setups, for CLSM, 218–219.Specifications, general, for scanner, 54.Specimen, general considerations, 192–197,

228, 361–362, 779. See also, Livingcells, Living embryo imaging.

fluorescent probes interactions, 361–362cytotoxicity, 362localization, 361–362metabolism, 361–362perturbation, 362

optical heterogeneity, 22, 23plants. See Plant cell imaging; Botanical

specimensSpecimen chambers. See Living cell

chambers.Specimen heating, in 2-photon, 539.Specimen holder, for scanning specimen, 9.Specimen preparation, for automatic 3D

image analysis, 319–321.image analysis, 319–321imaging artifacts, 320

Specimen preservation, general, 368–378.antibody screening on glutaraldehyde-

fixed specimens, 377evaluation, 371–374

cell height to measure shrinkage,371–373

defined structures, distortion, 373–374MDCK cell, stereo image, 373MDCK cell, vertical sections, 372

fixation/staining, 370–371fixative characteristics, 368–370

chemical fixatives, 369cross-linking fixatives, 369freeze substitution, 369microwave fixation, 369protein coagulation, 369

formaldehyde, 369–370, 373general notes, 374–378glutaraldehyde, 369, 370immunofluorescence staining, 371improper mounting, 376labeling thick sections, 376–377microwave fixation, 377–378mounting methods, 370–374

critical evaluation, 371–374mounting media, table, 377

pH shift/formaldehyde fixation, 370–371,373

refractive index mismatch, 377mounting media, table, 377

refractive index of tissue/organs, table,377

tissue preparation, 376triple labeling, 375–376

Specimen-scanning confocal microscope, 9.

Index 979

Speckle, from high-coherence sources, 8,84, 90, 130–132, 448.

Speckle microscopy, 13, 383, 385, 889.Spectra, emission.

arcs, 130black body, 136LEDs, 133solar, 127tungsten source, 153

Spectral accuracy, 662.Spectral bleedthrough (SBT), 185, 203–204,

664.in intensity-based FRET, 185

Spectral confocal image A. thalianaseedling, 175.

Spectral detector, 203–204, 662–663,666–667.

testing, 662Spectral discrimination, filter bandwidths,

44.Spectral imaging, 175, 382, 384.

table, 384Spectral leakage, inter-channel signal

imbalance, 185, 203–204.Spectral phase interferometry, for direct

electric field reconstruction(SPIDER), 115.

for pulse length measurement, 115,901–903

Spectral properties, of filters vs. angle, 49.Spectral resolution, of detection system,

203–204, 662–663, 666–667.Spectral response.

of CCD chips, 29, 234, 922of eye, 153PMT photocathodes, 29

Spectral transmission, objectives, plots,159–161.

Spectral unmixing, 190–192, 319, 361, 382,384, 386, 423–425, 431, 664–667,770, 905.

detectors for, 51, 667examples, 665–666limitations, 51, 382, 667overlapping fluorophore emission, 190,

319, 423–425, 664–667removing autofluorescence using, 667

Spectrofluorimetry, for FRET, 793, 795.Spectroscopic ruler, 765.Speed, in confocal imaging, 7, 11–12, 36,

41, 53, 142, 222–224, 235–236, 434,447, 450, 458, 460, 482, 526, 536,563–564, 748, 753–755, 784. Seealso, Temporal resolution.

4Pi-MMM, 563–564AOD, 55–56calcium imaging, 741CARS, 599–600, 604charge-coupled device cameras, 77–78,

142, 229, 231–235, 259, 647, 651,754–755, 885

data compression, 581–582, 586–588detector, in FLIM, 523, 558

disk-scanning confocal, 141, 216, 224,754

for display, processing, 803, 839, 841-842, 862

factors affecting, 235–236, 482, 496,753–754

of fixation, 370FRET, 795, 805galvanometer, 52–54, 211, 214high-content screening, 809–810, 813MMM, 551–555, 563–564need for, in living cell imaging, 222,

753–754rendering, 3D display, 831SPIM, 613, 678

Spermatocyte, crane fly, 15.Spherical aberration, 15, 34, 147–149, 151,

160, 192–197, 208, 241, 244, 247,330, 395, 404–413, 454–455, 463,466, 471, 480–481, 542, 629, 640,654–655, 657–658, 728, 772, 774,893, 903–904. See also, Aberrations,spherical.

blind deconvolution, 471, 480–481chapter, 404–413confocal microscopy performance, 654correction of RI mismatch, 192, 287, 411,

542correction of, figure, 145, 411–412,

654–655corrector, 92, 395, 398, 411, 477, 640,

654deconvolution, 463, 466, 468–469, 471,

480, 498–499, 658, 728, 784effect of specimen, 192–197, 418, 454,

747index mismatch. See Index mismatch

measurement, 145, 407, 455, 471,481, 492, 657

signal loss, 330, 389, 395, 413, 457, 661SPIDER, Spectral phase interferometry for

direct electric field reconstruction,115, 901–903.

Spill-over, between detection channels. SeeBleedthrough.

SPIM. See Selective plane illuminationmicroscopy.

Spinning disk, 3, 5–6, 11, 40, 141, 176, 216,223–224, 231–232, 235–236,260–265, 459–460, 464, 468,481–483, 783–784, 810–811. Seealso, Diskscanning confocalmicroscopy.

commercial, 907, 913, 915FLIM, 519–520, 522high-content screening, 810–811, 820MMM, 554, 558performance, 449–450systems for, cytomic imaging, 810vs. TPE imaging, in plant cells, 783Yokogawa CSU-10/22, 231. 915

Spinning-disk light scrambler, ground glass,8.

Spinning filter disk, digital projector, 590.Spirogyra, and depth of optical sections,

195.Spot scanning, to avoid coherence effects,

84.Spot size, full-width at half-maximum. See

Pointspread function, Full-widthhalf-maximum.

Square pixels, advantage of using, 62.Stability, 86, 102, 103, 136–139, 826.

algorithmic, 473arc sources, 136–137, 477argon-ion laser vs. krypton laser, 102disk scanners, 215of DVDs, 587dye. See Dyes; Bleaching from fiber-optic

coupler, 505–506galvanometer, 54halogen sources, 136–139, 346interferometer, 240–241, 267laser, 81, 85–89, 704

diode, 106, 108–109fiber output, 505helium-cadmium, (low), 103intensity, 85–87, 113, 116, 136, 477,

903measurement, 650–651pointing, 87, 903results, 86, 103structure, 82–85, 103thermal, 111wavelength, 106–108, 115, 118

mechanical, 39, 82, 85, 201, 267, 512,652

objectives, 146photostability, 363, 369, 690–702, 802.

See also, Dyes; Bleachingscan, 40, 638–639, 651shutter, CCD camera, 929thermal, 111, 219, 387, 389, 394, 539. See

also, Thermal variablesStage-scanning confocal microscope, 11.

piezoelectric scanners, 57, 708Staining, plants, 438, 774. See also, Dyes;

Livingcells; Botanical specimens;Plant cell imaging; Fluorophors.

calcofluor procedure, 438of plant tissues, 774

Standards, ISO (DIN) microscope design,156+.

Standing-wavefield microscope, 275.Starch granules, plant, 202, 420–421, 428,

432–433, 435, 703, 710–712, 715,719.

Static discharges, destroy semiconductors,109.

Statistical noise, in counting quantum-mechanical. See Poisson noise.

STED. See Stimulated emission depletion.Stem-cells, 623, 678, 762, 790, 813.Stem, plant, 168, 172, 180, 417–419, 421,

424, 430, 556, 707, 710–711,713–714.

980 Index

Stentor coeruleus, backscattered light image,168.

Step index optical fibers, 501–503.Stepwise discriminant analysis (SDA), 818,

820.Stereo Investigator, software, 282.Stereology, 316, 319.Stereoscopic image, about, 6–7, 9, 11, 154,

224, 298–299, 317, 396.biofilms, 880cheek-cell specimen, 23diatom, 640Drysophila, microtubules, 752embryo, 200fat crystal, polarization, 479neurons, 298, 314

Alexa stained, 330backscattered light images, 167eye, optic nerve, 481Golghi-stained, 298Lucifer-yellow, 314microglia, 396–398rat-brain neurons, 398transmitted light, 475

lung, 292MDCK cells, 373–374, 378Milium chromosomes, Fuelgen-stained,

298Paramecium, chromosomes, 298pea root, RNA transcript, 465platelet, high-voltage, EM, 848–849sea urchin, S. Purpuratus, 173, 198, 200skin, 298Spirogyra, 195tandem-scanning confocal microscope,

6Stereoscopic views, image processing and

display, 290, 292, 293, 295–299,451, 764.

color space partitioning, 297display, 293, 299interlaced fields of frame, 297movie projection, 838pixel-shift/rotation stereo, 297stereo images example, 298synchronizing display, 297

Stick objective, for in vivo confocal, 806.Stimulated emission depletion (STED)

microscopy, 3, 539, 561, 568,571–578.

axial resolution increase, 576breaking the diffraction barrier, 571–573challenges, 577compared to confocal, 575–576diagram, 573different approaches, 573dyes used successfully, table, 575OTF compared to confocal, 578outlook, 577PSF compared to confocal, 578RESOLFT, the general case, 572–573results, 576, 578triplet-state, 573

Stimulated emission of radiation, defined,82–83, 124.

Raman scattering, 167semiconductor, 106and stimulated-emission depletion, 573,

577STN, supertwisted nematic, 589.Stochiometry, ion kinetics, 741.Stokes field intensity, 595, 597.Stokes laser, in CARS microscopy, 595,

597–604.Stokes shift, 44–45, 268, 338, 341, 343,

443–447, 539, 542, 690, 759,792–793.

anti-Stokes,CARS, 550, 595–604defined, 44–45in fluorescence resonance energy transfer,

792+

large, in 2-photon, 539, 646of quantum dots, 694, 759size of fluorophores, 45

Storage, digital. See Data storage.Storage structures, plant, 435–436.

maize, image, 436Stray light, 58, 632, 904.

laser light, 632non-descanned detection, 904practical confocal microscopy, 632room light, 201, 632

Streak camera, FLIM detector, 520.Strehl ratio, measure of image sharpness,

247.S. purpuratus (Sea urchin), 173, 198, 200.

embryo, 173, 198, 200first mitotic division, 173image degradation, from top and

bottom, 198stereo-pairs of embryo, 200

Structural contrast, 188. See also, Harmonicsignals.

Structure, optical, 59, 68, 132–135.of light-emitting diodes (LED), 133of microscope sources, 132–135recognizing features in noisy images, 68chapter, 265–279

Structured illumination microscopy,265–279.

advantages/disadvantages, 265computing optical sections, 268–270vs. confocal microscopy, 265degree of spatial excitation modulation,

268–270absolute magnitude computation,

268–269homodyne detection scheme, 268–269max/min intensity difference, 268scaled subtraction, 269–270square-law detection, 268–269synthetic pinholes, 268, 269

experimental considerations, 265–268illumination masks for, 266light source for, 267

pattern generation, 266–268schematic setup, 266

nonlinear, 276resolution improvement, 270–276

Fourier-space, 270–271linear image reconstruction, 271Lucosz’s formulation, 273Moiré effects, 270–271photobleaching, 275reconstruction steps/results, 272standing-wavefield microscope, 275test results, 274thick samples, 274, 275, 278–279

Subcellular location features (SLF) inautomatic image analysis, 819–820,822–824, 828.

2D, 819–8202D SLF feature descriptions table, 8193D SLF, 822–823

test results, table, 824Subcellular location tree (SLT), 825.Subpixel deconvolution, 478–479.Subresolution beads, 655–656. See also,

Beads.Sun, microscope light source, 126–127, 131,

135.spectrum, 127

Superficial optical sections, living embryo,748.

Supertwisted nematic (STN), 589.Surface imaging microscopy (SIM),

607–608.mouse embryo, 608setup, 608

Surface mount device (SMD), for LED, 133.

Surface orientation, affects reflected light,181.

Surface structures, distortion, 197.Surface topography, maximum intensity,

180.Surfaces, of interference filters, 47.Suspension-cultured cells, 189, 429–430.

bacteria, 876, 878image, 430frozen, 854

Swept-field confocal microscope, 238.Synchrotron, wide-spectrum light source,

135+.Synthetic pinholes, in structured-

illuminationmicroscopy, 268, 269.

images, 269SYTO, 396, 874–876, 879–885.

TTagged image file format. See TIFF.Tandem-scanning confocal microscope

(TSM), 2–6, 11, 13–15, 39–40, 141,167, 215–216, 223–224, 228–229,447.

comparison with other confocals, 13–15

Index 981

description, 6, 141, 215–216, 228–229development, 5–6evaluation, 215, 216observing ciliate protozoa, 141rate of data acquisition, 11real-time imaging of tooth, 167sources of vibration, 39–40viewing color/depth-coded, real-time,

stereoimages, 154, 304

Tapetum, plant, 433, 434, 779.TEC, Thermo-electrically cooled, see Peltier

cooling.Telan systems, 129, 157.Telecentric plane, 208–209, 211.

conjugate, 208–209effect of angular deflection in, 211

Telecentricity, 207, 214.of closely-spaced scan mirrors, 214defined, 207Tellurium oxide (TeO2), for use in AODs,

55TEM. See Transmission electron

microscope.TEM. See Transverse electromagnetic

modes.Temperature, 29, 56, 133, 135–136, 856,

885. See also, Thermal variables.Temperature tuning, of diode lasers, 108.Temperature effects on high NA objectives,

248+.Temporal aliasing, 39, 41, 391, 836–837,

839.Temporal coding, 299–300.Temporal coherence, 7–8, 82–85, 131.

defined, 84Temporal dispersion, 502. See also, Pulse

broadening.Temporal displays, 292–293, 297, 836.Temporal experiments, biofilms, 885–886.Temporal pulse behavior, pulsed laser, 111.

See also, Pulse length measurement;Pulse broadening.

Temporal resolution, 12, 24, 36–38, 41,221–222, 322, 334, 386, 391, 399,458, 558, 577, 618, 620, 622, 651,667, 730, 737, 746, 772, 784, 801,809. See also, Fluorescence lifetimeimaging (FLIM).

of photodetectors, 263Temporal signals, 162, 286, 331, 383.“Test drives,” for living embryo imaging,

752.TFT. See Thin-film transistor.Tetracysteine, labels, 221, 348, 359, 853.Thalamocortical slice protocol, 724.Thermal lensing, pulsed lasers, 109, 113,

543.Thermal variables, 219, 856.

active medium, lasers, 81of AODs, 56–57arcs, peak emission wavelengths, 129automated confocal imaging, 810

cell chambers, 117, 386–389, 394, 727,790, 810, 814, 885–886. See also,Cell chambers cooling, 108, 133

cryo preparation for EM, 856–857on detectors, 29, 252, 256–257, 495drift, 16, 115, 219, 386, 567, 489, 652

compensating, 396, 732on dye labeling, 359, 361, 738–739effects of anti-bleaching agents, 694effect on bleach rate, 696–689effect fiber pinhole size, 506fiber-optic, pol-preserving fiber, 503filament spectra, 135–136fixation, 369–372, 375, 377incandescent lamp emission, 135–136laser cavity, 34, 82, 85–88, 107, 109, 111,

541of LED, 133, 136–138

brightness, 133lensing, in pulsed lasers, 109, 113, 543and light-source output, 136, 138, 650noise signal, 254, 257, 232–234,

261–262, 495, 660, 734, 921, 924,925

on objective lenses, 248–249in photography, 71properties of ice, 856properties of optical materials, 158,

248–249and photomultiplier tube, (PMT), 29on refractive index, 15, 56, 145, 411

immersion oil, 148–149, 248–249, 411

retinal exposure, 117–118sensors, 255–256, 727solid-state laser, 86, 108specimen damage, 84–85, 139, 685specimen heating, 539, 545, 681, 685,

904temperature tuning, laser, 108, 115thermomechanical effects, 685time constant, 38

Thermo-electrically-cooled, see Peltier-cooled.

diode lasers, 85, 107–108, 111, 117THG. See Third harmonic generation.Thick samples, 274, 275, 278–279. See also,

Living embryo imaging; Brain slices;Biofilms.

background, 278structured illumination, 274, 275,

278–279close focus region, 279distant focus region, 279in focus region, 278number of collected photons, 279

Thin disk lasers, 109–110.Thin Laser Light Sheet Microscope

(TLLSM), 672. See also, SPIM.Thin-film transistor (TFT), 589.Third harmonic generation (THG), 90,

166–167, 179–180, 188, 428, 435,550, 705–718.

CARS, 596–597contrast mechanism, 166–167deposits no energy, 361detectors for, 421, 706–708

table, 707double-pass detection method, 166–167intracellular inhomogeneities tracked,

90light attenuation spectra, 706light sources, 706–708to make more laser lines, 109, 114mechanism, 705microspectroscopy, 421MMM, 551, 559non-linear optical microscopy, 705optical sectioning, 704optically active animal structures,

714–717collagen mat, polarization microscopy,

717mouse zygote spindle, 717structures producing THG, table, 715zebrafish embryo, 716, 718

optically active plant structures, 710–714cell walls, 438Commelina communis, 712Euphorbia pulcherrima spectrum, 710maize, emission spectrum, 710, 711,

713maize, polarization microscopy, 711maize, stem section, 714phytoliths, polarization microscopy,

720potato, 712Pyrus serotina spectrum, 711rice leaf, image, 712, 715, 719

photon interactions, 179pulsed lasers suitable, table, 706STED, 577structural contrast, 188

Three-decibel point (3dB), for bandwidth,59, 65.

Three dimensional cell pellet, 815.Three dimensional microscopy, 766, 771,

804+.future perspectives, 804–805living embryos, 766of plant cells, 771

Three dimensional projections, embryo, 763.Three dimensional segmentation, plant,

776–778.Three-channel confocal microscopy.

with 4 recombinant proteins, 190assays for, 814

Three-dimensional diffraction image, 4, 147,407, 455, 463, 471, 491.

Three-dimensional micro-array assays,815–816.

Three-dimensional reconstruction, 775–776,778, and Chapters 14 and 15.

plant imaging, 775–776A. thaliana, 778Equisetum, 774

982 Index

Three-photon excitation (3PE), 88, 415, 447,535, 550–552, 555, 558, 647, 680,709, 876.

absorption cross-section, 680damage, 682, 686fiber-optics, 507resolution, 447setup, 708–709

TIFF (Tagged image file format), 580.Tiled montage, 851, 858.Tiger, ECDL laser system, 90.Time correlated single-photon counting

(TCSPC), 518, 520–523, 526.for lifetime imaging, table, 526FLIM, 520–523FRET-FLIM, 186schematic diagram, 521

Time multiplexing, of adjacent excitationspots, to reduce flare in MMM,553–554.

Time-gated detection, FLIM, 522–524, 526,528+.

diagram, 522FLIM methods compared, table, 526FLIM, image, 528–529

Time-lapse imaging, 136, 222, 354,382–384, 392–399, 652, 773,885–886.

Amoeba pseudopod, 191confocal of plant cells, 773high-content screening, 812illumination stability, 136image analysis, 286, 320, 333, 732–733mechanical stability, 219microspectrometry, maize damage,

424–426rectified-DIC, of platelets, 846SPIM, 613table, 384three-dimensional plus time, 222two-dimensional plus time, 222

Time-lapse recordings.Amoeba pseudopod, 191Ascaris sperm, 846biofilms, 885brain slices, 725, 727, 729, 732–733embryos, 676, 749, 752, 759, 761meristem growth, 430plant roots, 781, 784rectified-DIC, of platelets, 846two-photon microscopy, 10

TIRF. See Total internal reflectionfluorescence.

TIRM, 177–179, 477.Tissue specimens, introducing the probe, 360.Titanium:sapphire laser (Ti :Sa), 82, 84–86,

88–91, 94, 100–103, 105, 107, 109,111–112, 114, 123–124, 165, 346,358, 415, 423–424, 459, 541, 550,551, 645–647, 688, 706–708, 713,727, 750, 756, 759. See also, Lasers,titanium: sapphire and Ultrafastlasers.

4Pi, 563–564, 567brain slices, 731CARS, 599compare to other fast lasers, 112–113Cr :Fosterite, femtosecond pulsed laser,

109, 114, 415, 541, 706–709,712–714

embryos, 750, 756, 759, 731, 764emission stability, 86four-level vibronic model, 82, 109maintenance, 116multi-photon excitation, 541and OPOs, 114–115plants, 415, 423–424, 706–708, 713–714,

717, 781–783popular models, specs, table, 120STED, 575ultrafast, 112–113URLs, 124

TLB. See Transmitted light bright-field.TLLSM. See Thin Laser Light Sheet

Microscope.Tobacco, 116, 189–190, 430, 693.

smoke, not around lasers!, 116suspension-cells,

birefringence, 189–190GFP expressing cells, 430photo-bleaching, 693

“Toe” photographic response, defined, 71.Tornado mode, SIM scanner, 54.Total fluorescence signal, 742.Total internal reflection fluorescence

microscopy (TIRF), 90, 160,180–184, 223, 477, 801.

blind deconvolution, 477vs. confocal image, 184contrast, 180–184cytoskeleton, image, 183FRET, 801limits excitation to single plane, 223objectives, for epi-TIRF, 161

Total internal reflection microscopy (TIRM),177–179, 477.

blind deconvolution, 477evanescent wave generation, 178

TPE. See Two-photon excitation.TPEM. See Two-photon excitation

microscopy.Trade-offs, 36, 68, 78–79, 221, 224,

644–648, 747–748, 825.beam power, visibility/damage, 693blind deconvolution, 483, 488, 499compression algorithms, 581, 840confocal endoscopes, 508when digitizing, 68, 78–79embryo specimens, 747–748high-content screening, optimal

clustering, 825living cells, 381, 693micro-CT, dose/resolution, 616MRM, time/resolution, 622and pinhole size, 265, 267processing speed/segmentation, 301

speed, S/N, sensitivity and damage, 221,224, 232, 556, 644–648

SPIM, resolution and number of views,613

Transcriptional reporters, embryo analysisand, 748, 755–756.

FluoroNanoGold, 854mRNA, 316–317, 465plants, 773, 781NF-kB, 814

Transfection buffer, electroporation, table,802.

Transfection, cellular, 756–758, 790, 791.brain slices, 722, 724–725, 730–731

Transfection reagents, for chromophores,358, 360, 362, 556, 682, 790–791,795, 803.

2-OST-EGFP, 566COS7, 693EB3-GFP, 183for FRET, CFP/YFP, 795–796, 798,

801–802GaIT-EGFP, 566GFP-MusculoTRIM, 184ligand binding, 348

Transfer function, implications for imagecontrast, 164–165. See also, CTF.

Transient permeabilization, 359, 373, 375.Trans-illumination, absorption contrast, 166.Transistor-transistor logic (TTL), 259.Transit time spreads (TTS), 527.Translational fusions, 756, 757. See also,

Transfection agents.subcellular specific protein distribution,

756Transmission, 33, 49, 159, 225, 231, 804.

AOBS, 57contrast, 163–164disk-scanning micro-lens array, 223–226,

227–229, 231, 235dispersion, 683of filters. See Filters

linear vs. log plots, 44–49of glass fibers, 501–505illuminator, 201, 127–128losses due to refractive optics, 33, 217

table, 217of objectives, 154, 158, 159–161, 641

relative, measurement, 26, 34, 36table, transmission, 158, 159–161

of plant tissue, spectra, 416, 422of Polaroid materials, 85SHG signal detection, 707–709, 729–730by small pinholes or slits, 225

Transmission electron microscope (TEM),846.

correlated LM-TEM images, 852–855,857–859

stereo images of platelets, 848–849Transmission illuminator, ghost images,

201–202.Transmission intensity, specimen thickness,

164.

Index 983

Transmittance, optical system, measured,25–26.

table, 217Transmitted light brightfield, 468, 472–473,

477.blind deconvolution, 472–473, 477

Transparency, lighting models, 309–312.Transverse electromagnetic modes (TEM)

laser, 83.Trends, in laser design, 118.Triple-dichroic, 33, 46, 48, 217–218, 678,

783.light loss due to, 33performance, 46–48

Triplet state, 103, 338, 339–342, 348,362–363, 390, 516–518, 573, 646,684, 691–693, 697, 698, 704, 852.

saturation, 339, 573as a RESOLFT mechanism, 573

Triton X-100, 730, 852.formaldehyde fixation, 370–372,

375–377True color, 291.TSM. See Tandem-scanning confocal

microscope.TTL. See Transistor-transistor logic.Tube length/chromatic corrections, table,

157.Tunable lasers, 91, 103, 107, 109, 120.

broadband, table, 120continuous wave dye, table, 91diode, emerging techniques, 107solid-state, 106, 109solid-state ultrafast, 103

Tungsten carbide electrodes, radiance,137–138.

Tungsten halogen source, 132, 137, 153.Turnkey ultrafast laser systems, 118.Tutorials, lasers by level, 124.Tweezers, optical, 89–90, 110, 218, 383,

385.setups for integration, 218single-longitudinal-mode fiber laser for,

110trapping wavelength, 89–90

Two-channel confocal images, 175–177,177, 193, 425, 522.

A.thaliana, epidermal/mesophyll cells,193, 425, 431–432, 434–436

Amoeba pseudopod, 169colocalization, 667display, 311, 841FLIM, 522harmonic images, 714–716

mouse muscles, 716montaging, 331neurons, 332

microglia, 396–398eye, optic nerve, 481Golghi-stained, 298Lucifer-yellow, 314rat-brain neurons, 398transmitted light, 475

of peony petal, cytoplasmic, 175–176rat intervertebral disk, 310–311of zebrafish embryo, 177

Two-dimensional imaging, 60, 222,397–398.

time lapse, 222, 397–398Two-photon fluorescence excitation (2PE),

156, 160, 218, 535, 536, 750,778–783.

chapter, 535–549chromatic correction for, 156for plant cells

advantages of, 778–779cell viability, 779–781vs. confocal microscopy, 779dyes, 782of green fluorescent protein, 782–783pitfalls, 782of thick specimens, 779in vivo, 781

special objectives for, 160visible and ultraviolet dyes, 218

Two-photon microscopy, 10–12, 195, 357,535–549, 690, 697, 900–905. Seealso, Multi-photon excitation; Multi-photon microscopy

autofluorescence, 545basic principles, 535of biofilms, 882–885bleach planes, in fluorescent plastic, 193,

194caged compounds, 544calcium imaging, 545chromophores, 543

2-photon absorption, 543diagram, 540detection, 538, 541

descanned, 542non-descanned (whole area) detector,

541stray light, 904

fluorescence, shadowing, 195group delay dispersion, 5443laser. 540–541

alignment, 900–904monitoring, 901–903mounting, 541power level, 903–904safety, 117–118, 839, 900,

903–904living cell studies, review, 544–545living animal studies, 545minimize exposure during orientation,

905mirror scanning, 543optical aberrations, 542photobleaching, 690, 697practical tips, 900–905

beam alignment, 901bleed-through, 904choice of pulse length, 537, 903

pulse length, 109, 112, 115, 507, 537,538, 902–903

specific specimens, see specimens byname imaging multiple labels,904–905

neurolucida protocol, 731resolution, 539and speed, 12vs. spinning disk imaging. in plant cells,

783stray light and non-descanned detection,

904theory, 535, 537wavelengths, 538–541, See also,

Botanical specimens

UUBC 3D living-cell, microscopy course,

174, 183, 184, 190, 205, 364, 430,435, 439, 805–806.

Ulbricht sphere, for measuring light, 140.Ultrafast imaging, two dimensional, 222.

3D, 235Ultrafast lasers, 88, 101, 103, 112–114.

Cr :Fosterite. 109, 114, 415, 541, 706,707–709, 712–713

diode-pumped solid-state (DPSS), 112distributed feedback (DFB) diode laser,

113fiber, 113–114

table, 101fiber-diode, mode-locked, 113Nd :YAG, 88–89, 91, 95, 97, 103,

107–109, 111, 113–115, 117, 218,245, 514, 680, 798

Nd :YLF, 89, 98, 100, 103, 109, 112–114,750, 760–761

Nd:YVO4, 89, 95, 100, 103, 107–109,111, 113–114, 541

solid-state, tunable, 103spectrum, 44titanium: sapphire, 112–113. See also,

Laser, titanium: sapphire; Titanium-sapphire laser

Ultrafast pulses, delivery by fiber optics, 88,507.

dispersion losses, 502Ultraviolet (UV), argon-ion laser lines, 85,

87, 90, 102, 339, 346.other UV lasers, 111–117use for micro-surgery, 218–219

Ultraviolet (UV) confocal microscopy, 109,174, 195, 571.

absorption, 707, 713autofluorescence, 431–432, 434, 544CCD response, 29, 255, 459, 921–922correct imaging with planapochromats,

14, 154damage, 212, 290, 439, 544, 680, 686,

903disk-scanners, 229DNA-dyes, 782, 874. See also, DAPI;

Dyes GFP excitation, 798, 873high-content screening, 811ion-imaging, 346, 383, 529, 738, 742

984 Index

Ultraviolet (UV) confocal microscopy(cont.)

multi-photon excitation, 535, 538, 544,559, 646, 706, 905

photoactivation, 759safety, 117–118, 839, 900, 903–904simultaneous with DIC imaging, 846,

850as source of stray signal in PMT

envelopes, 257Ultraviolet performance of objective lenses,

154, 159–161, 706.Ultraviolet widefield light sources, 132, 136,

139, 143, 226, 542.table, 226

Ultraviolet transmission of optical fibers, 88.

Ultraviolet (UV) light, effects produced bymultiphoton intrapulse interference,88.

Ultraviolet scanning light microscope, 6–7.Uncaging, multi-photon microscopy, 383,

385, 545, 693, 760–764. See also,Photoactivation.

Unconjugated bodipy/ceramide dyes, 760.Under-sampling, 79, 635, 640, 652, 662,

831, 833, 836, 839, 841.example, 640uses, 68

Uniformity, of light source, 127–129.Unit image body, 3D Airy figure, 147.Upright vs. inverted microscope, 140, 157,

217, 230, 413, 722, 727, 870–872.Unmixing. See Spectral unmixing;

structured illumination.Up-conversion, fiber lasers, 110.

doped ZBLAN, 110dual-ion doped, 110

UV. See Ultraviolet.

VVacuum avalanche photodiode (VAPD), 31,

254, 255.definition, 254schematic, 31, 255

VAPD. See Vacuum avalanche photodiode.Vertical-cavity semiconductor diode laser

(VCSEL), 108.Vibration.

compensation, 732from cooling water, 84, 102, 499of disk scanner, 753causing distortion, 16, 39–41, 166, 201of galvanometer mirrors, 40, 201high-frequency, of acousto-optic devices,

55, 84isolation, 85, 201, 219, 541measurement, 30–41, 652of mechanical shutters, 929of objective lens motion, 754optical fiber isolation, 505, 507of optical fiber scrambler, 8, 84, 131

Vibronic laser, Ti :Sa four-level, 109.

Video, 2, 4, 5–7, 11–14, 17, 37, 52–53,61–62, 88, 219, 237, 261, 263, 346,372, 430, 451, 505, 539, 554, 556,589–590, 593, 604, 860, 885.

confocal, 25, 237, 914impact on light microscopy, 5–7, 14

results, 14signal, 258–259

Video-enhanced contrast microscopy,imaging small features, 14, 68.

Vignetting, 210–211, 229, 245–247, 492,541.

objective, off-axis performance, 245–247Visibility, and signal-to-noise ratio, 37–38,

68. See also, Rose Criterion.Visilog/Kheops, software, 282, 301–302,

312.Visitech, confocal manufacturer,

descriptions, 88, 119–120, 226, 237,908.

VT eye, 119–120, 908, 914VT Infinity, 119–120, 908, 914

Visual cortex, identification of primary, 724.Visual observation, magnification for, 146.

non-linearity, 72–73Visualization, 280, 282–283. See also,

Multidimensional microscopyimages; Rendering.

definition, 280, 292software packages for, table, 282–283

Vitrea2/Voxel View, software, 282, 335.Volocity (software), 281, 236, 282, 295,

299, 312, 757, 762–764.VolumeJ, software, 282, 304, 764.VolVis, 281–282.VoxBlast, 283, 301–302, 309, 312.Voxel, defined, 20.Voxel rendering, speed, 290.Voxx, software, 283, 377, 764.

WWAD. See Whole-area; Non-descanned

detection.Water, as immersion medium, 409, 410.

refractive index mismatch, table, 409, 410two-edge response curves, 410

Water-coverslip interface, sphericalaberration generated at, 147.

Water-immersion objectives, 15, 23, 36,141, 148–149, 154, 190, 235,241–242, 247, 261, 377, 386–387,389, 395, 411–412, 513, 542, 552,556, 562, 567–568, 584, 654–656,708, 727–728, 737, 747, 772. Seealso, Spherical aberration.

4Pi, 562, 567–568advantages, 149biofilms, 870, 872brain slices, 727–728, 730, 737chapter, 404–413correction-color/flatness/transmission,

154deep imaging, 395

dipping objectives, 161, 209, 411, 429,568, 613, 727, 737, 870, 872

in fluorescence ion measurement, 737ion measurement, 737living cells, 386–387, 389, 395, 398performance measured, 47, 655–656plant cells, 429, 433, 772STED, 576transmission curves, 159–161use and limitations, 15

Watershed algorithm, 322–325, 777, 822.for segmentation, plant cell images, 777

Wave optics, 4, 10.for calculating axial resolution, 4, 146,

154Wavefront error, 217.

lower, with hard coatings on filters, 45Wavelength, 24, 28, 43–51, 62, 88, 107,

114–115, 118, 129–130, 135–139,165–166.

calculation of Forster radius, FRET, 793and CCD coupling tube magnification, 62filters for selecting, 43, 44, 88in multi-photon lasers, 165–166. 415, 750multiple, dynamic embryo analysis, 756of non-laser light sources, 129–130,

135–136and optimal zoom setting, 24vs. pinhole size, 28selecting, with interference filter, 88,

165–166stability, in non-laser light sources,

137–139tunability, of lasers, 107, 109.

Wavelength expansion, non-linear, 114–115.Wavelength ratioing, 346. See also, FRET;

FLIM.Wavelength response, chromatic aberration,

663.Wavelength-selective filters, 43–51, 88.Wavelength-tunable lasers, summary, 107,

113, 116, 118, 550.Wavelet compression, 581–584.Wavelet de-noising protocol, 733–734,

819–820.Waxes, plant, 420, 428, 434–435, 714–715.Website references, 123.

2 photon excitation spectra, 546, 727,729, 782

brain slices, 727CCDs, 76, 234, 927, 931components, 58confocal Listserve, 390, 901deconvolution, 495dyes, 221, 343–344, 782fluorescent beads, 653FRET technique, 185, 803high-content screening systems, 811image management, 865lasers, 104, 115, 120, 123–125live-cell chambers, 388–389, 870movies related to book, 235, 392muscles, 237

Index 985

non-laser light sources, 138, 143plants, 769safety, 117–118, 839, 900, 903–904software, 282, 376, 594, 734, 762, 764,

776, 777, 820, 824, 827, 831–833,844, 845, 864–862, 865–867, 869

SPIM, 672Wedge, compensator, 566–567.Wedge, rotating, for light scrambling, 84,

131.Wedge error, in interference filters, 45–46,

151, 211–212, 630.in traditional filters, 45

Wedged fiber-optics, reduce reflections, 85.Well-by-well data, 817.WF. See Widefield.WFF. See Widefield fluorescence

microscopy.White light continuum lasers, 88, 109, 113

continuum, 88, 109He :Cd, 113.

Whole-area and external detection, 541–542.See also, Non-descanned detectors.

Whole-cell patch pipet delivery, 360,726–727.

Widefield deconvolution, 751–753, 785. Seealso, Deconvolution.

botanical specimens, 785for living imaging, 751–753

Widefield (WF) fluorescence microscopy, 3,22–23, 26, 172–173, 219, 453–467,518. See also, Epifluorescencemicroscopy, Deconvolution.

compared to confocal, 453–467, 644–647CCD/confocal comparison, 458–459,

465same specimen, 465, 482

compared to structured illumination, 274deconvolution, imaging living cells, 23,

392deconvolving confocal data, 461–464,

466fluorescence detection, 459–460fluorescence excitation, 459fluorescence lifetime imaging, 518gain-register CCDs, 460–461images utilizing out-of-focus light, 26imaging as convolution, 453–457imaging thin specimens, 172–173integration of fluorescence intensity,

459interaction of photons with specimen,

22–23light-emitting diode sources, 136limits, linearity/shift-invariance, 457, 490,

564model specimens, 461noise, 459–463optical sectioning schematic, 469optical tweezers/cutters, 219, 89, 383, 385out-of-focus light, 461point-spread function, 453–457, 459–463resolution, 3

sensitivity, 459–463single point images, 454pros/cons, 644–648

table, 459temporal resolution, 458

Wiener filtering, 494, 496. See also,Gaussian filtering.

image enhancement, 496image restoration by, image, 494

Windows software, for automated confocal,810.

WinZip, 580.Wollaston prisms, DIC, 156, 468, 473, 475.

See also, Nomarski; DIC contrast.Working distance (WD) of objective lenses,

5, 9, 129, 145, 154, 157, 198, 249,511, 568, 598, 634, 673, 678,727–728, 747, 774, 779, 781, 872.

table, 157–158WORM disks (write once, read many), 586.

XXenon arc lamps, 44, 132, 137–138, 144.

iso-intensity plot of discharge, 132pulsed-operation, 137–138shapes of electrodes, 132spectral distribution, 144super-pressure, spectrum, 44, 136

explosion hazard, 136wavelengths available for detection, 44

Xenon/iodine fill arc, radiance, 137–138.Xenopus laevis, 13, 610, 746, 748–753.

blastomere, 757confocal/multi-photon comparison, 750embryo

viewed with confocal, 748–753viewed with OCT, 610, 749

embryo viewed with MRM, 623–264in situ imaging, 746, 748oocyte wound closure, 749

X-Y resolution, confocal/widefieldcompared, 36.

YYellow fluorescent protein (YFP), 221–222,

429.FRET pair with CFP, 791–803

YFP, 221–222, 429Yokogawa disk-scanning confocal system, 6,

12–13, 16, 216, 224–226, 231,234–237, 458, 754.

CSU-10/22 model, 223, 231, 236, 915with EM-CCD, 234, 237, 755

high speed acquisition, 11, 220, 222–226,229, 231, 458, 667, 754, 784

results, 236–237, 755, 783vibration, 16

Ytterbium tungstate (Yb :KGW) laser, 108.

ZZBLAN up-conversion glass fiber, 110.Z-buffering, 304–305.Z-contrast, in confocal microscopy, 180.

Zea mays. See Maize.Zebrafish, 174, 176, 761.

GFP image, 176, 176autofluorescence, 174

pancreas expressing DsRed, 176scatter labeling/lineage tracers, 761

Zeiss, confocal manufacturer, 212, 214, 217, 226, 231–232, 655, 771,916–917.

510 META confocal microscope, 655,908, 916

Achrogate beam-splitter/LSM 5-Live, 50,119–120, 212, 231–232, 916

Axioimager system, 217fluorescence correlation spectrometer

(FCS), 383, 385, 602, 801, 803, 805,917

HBO-100 source, self-aligning, 134–135high-content screening, 811LSM 5-Live line-scanning confocal

microscope, 50, 51, 231–232, 237,784, 908, 916

META confocal spectral detector, 51,119–120, 161, 202, 660, 663, 796,916.

mini-PMT arrays, 51, 667FRET, 706tests, 663

objectives, advantages of, 155–156Infinity Color-corrected System, 155,

217plan objectives, table, 152transmission specifications, 161

tube length conventions, 157, 239working distance of objectives, table,

158Zernike moments, 247–249, 818–820.Zernike polynomial fit, 245–247.

table, 247wavefront aberration function, 247

Zinc selenide (ZnSe) diode lasers, 106.Zirconium arc lamps, 136, 141.

spectrum, 136Zone System (Ansel Adams), 71–72.Zoom magnification, 11, 24, 37, 63–64, 66,

70. See also Magnificationoptimal, 24optical vs. electronic bandwidths, 70relationship to area scanned, 63

Z-position and pinhole/slit size, 227.Z-resolution, 3–4, 22, 36, 149–150, 224,

225–228, 563, 752. See also, Axialresolution.

4Pi microscopy, 563in confocal fluorescence microscopy, 36effect, of fluorescence saturation, 22improvement, 752of pinhole disks, 224in STED, 576

Z-scanners, evaluating, 215.Z-sectioning, imaging brain slices, 729.Z-stack, 23, 754.

of images of cheek-cell specimen, 23speed acquisition constraint, 754

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