HANDBOOK OFBIOLOGICAL

CONFOCAL MICROSCOPY

James B. Pawley

Springer

987-0-387-25921-5

Springer Science+Busine. Media. LLC) 2006.

Preface to the Third Edition................................................. vii Resolution: How Much Is Enough? ...................................... 36Preface to the Second Edition ...............................................ix Can Resolution Be Too High? ................................................... 36Contributors.........................................................................xxv Limitations Imposed by Spatial and Temporal

Quantization.............................................................................37Practical Considerations Relating Resolution to

CHAPTER 1: FOUNDATIONS OF Distortion 39

CONFOCAL SCANNED IMAGING IN LIGHT Conclusion .............................................................................41

MICROSCOPY

Shinya [none CHAPTER 3: SPECIAL OPTICAL ELEMENTS

Light Microscopy..................................................................... 1 Jens Rietdoif and Ernst H.K. StelzerLateral Resolution.......................................................................... 1

Introduction...........................................................................43Axial Resolution............................................................................3Regulating the Intensity........................................................43Depth of Field

" " " " " " " " " " " " " "4

Wavelength Selective Filtering Devices ...............................43Confocal Imaging ....................................................................4Selecting the Wavelength of the Illumination andI mpact of Video.......................................................................5

Nipkow Disk..................................................................................5 the Detected Light...................................................................44....Separating the Light Paths ......................................................... 44Electron-Beam-Scanning Television............................................6 Conventional Filters 45Impact of Modern Video.............................................................. 7 Interference Filters

" " " " " ' " " " ' " " 45Lasers andMicroscopy ' ' ' ' ' ' ' ' ' ' ' 7 Dichroic and Polarizing Beam-Splitters ................... 50Holography..................

' ' ' ' ' ' ' ' ' ' Filters and Dispersive Elements for Multi-ChannelLaser Illumination .........................................................................8 Detection.................................................................................. 5ILaser-Illuminated Confocal Microscopes ................................... 9 Mechanical Scanners 51Confocal Laser-Scanning Microscope 9 Galvanometer Scanners.............................................................. 52Two- and Multi-Photon Microscopy.................................... 10Is Laser-Scanning Confocal Microsopy a General Specifications .................................................................54

Acousto-Optical Components ............................................. 54Cure-All?

. 11Speed of Image or Data Acquisition.......................................... 11 Acousto-Optical Deflectors 56

Acousto-Optical Modulators...................................................... 56Yokogawa Disk-Scanning Confocal System .............................12Acousto-Optical Tunable Filters ................................................ 56Depth of Field in Phase-Dependent Imaging ............................ 13Acousto-Optical Beam-Splitters .................................................56Other Optical and Mechanical Factors Affecting

Electro-Optical Modulators ..................................................57Confocal Microscopy.........................................................13Piezoelectric Scanners .................................................................57Lens Aberration............................................................................13

Polarizing Elements 57Unintentional Beam Deviation...................................................15Contrast Transfer and Resolution in Confocal

Removing Excess Light..........................................................58

Versus Non-Confocal Microscopy....................................16Summary................................................................................ 16 CHAPTER 4: POINTS, PIXELS, AND GRAY

LEVELS: DIGITIZING IMAGE DATA

CHAPTER 2: FUNDAMENTAL LIMITS IN James B. Pau'Iey

CONFOCAL MICROSCOPY Contrast Transfer Function, Points, and Pixels ....................59Pixels, Images. and the Contrast Transfer Function 59James B. Pawky Digitization and Pixels.......................................................... 62

Introduction...........................................................................20 Digitization of Images................................................................ 62What Limits?................................................................................20 How Big Should a Pixel Be? Sampling andCounting Statistics: The Importance of n.................................20 Quantum Noise................................................................. 63Source Brightness........................................................................21 The Nyquist Criterion........................................................... 64Specimen Response: Dye Saturation .........................................21 Estimating the Expected Resolution of an Imag e ....................65A Typical Problem.......................................................................24 The Story So Far......................................................................... 68

Practical Photon Efficiency ................................................... 24 Reality Check?....................................................................... 68Losses in the Optical System.....................................................25 Is Over-Sampling Ever Wise?....................................................68Detection and Measurement Losses ..........................................28 Under-Sampling?.........................................................................68Where Have All the Photons Gone?..........................................33 Digitizing Trade-Offs 68

Nyquist Reconstruction: "Deconvolution Lite "................................68 Modulated Diode Lasers............................................................112

Some Special Cases.....................................................................70 Diode Pumped Solid State Laser in Pulsed Mode ..................112

Gray Levels, "Noise, " and Photodetector Ultrafast Diode Pumped Solid State Lasers ............................ 112

Performance ...............................................................................71 Titanium-Sapphire and Related Ultrafast Lasers .....................I I2

Optical Density ............................................................................ 71 White Light Continuum Lasers .................................................113

The Zone System: Quantified Photography ..............................7 1 Ultrafast Fiber Lasers .................................................................1 13

Linearity: Do We Need It?......................................................... 72 Wavelength Expansion Through Non-Linear

Gray Levels in Images Recorded Using Techniques.................................................................................114

Charge-Coupled Devices: The Intensity Spread Second and Higher Harmonic Generation: SHG,

Function...................................................................................... 74 THG, FHG Label-Free Microscopy.....................................114

What Counts as Noise?...............................................................74 Sum or Difference Mixing........................................................ 114

Measuring the Intensity Spread Function ...............................75 Optical Parametric Oscillators and Optical Parametric

Calibrating a Charge-Coupled Device to Measure Amplifiers................................................................................114

the ISF......................................................................................75 Pulse Length Measurement ........................................................115

"Fixed-Pattern" Noise .................................................................76 Maintenance..................................................................................115

Gain-Register Charge-Coupled Devices.................................76 Maintenance of Active Laser Media ........................................ 115

Multiplicative Noise ....................................................................77 Maintenance of Pumping Media ...............................................116Trade -Offs.......................................................................................79 Maintenance of the Optical Resonator .....................................116

Maintenance of Other System Components ............................116Troubleshooting............................................................................117

CHAPTER 5: LASER SOURCES FOR Safety Precautions.......................................................................117CONFOCAL MICROSCOPY Beam Stops..................................................................................118

Curtains........................................................................................118Enrico Gratton and Martin J. vandeVen Laser Goggles ............................................................................. 118Introduction................................................................................... 80 Screens.........................................................................................118Laser Power Requirements.........................................................80 Exposure Effects, Warning Signs, and Interlocks...................118The Basic Laser ..............................................................................81 Infrared Paper..............................................................................118

Principle of Operation .................................................................82 Conclusion......................................................................................118Pumping Power Requirements...................................................82Laser Modes: Longitudinal (Axial) and

Transverse................................................................................82 CHAPTER 6: NON-LASER LIGHT SOURCESPolarization.................................................................................. 83 FOR THREE-DIMENSIONAL MICROSCOPYCoherent Properties of Laser Light 83

Andreas Nolte, James B. Pawley; and Lutz HroringPhase Randomization: Scrambling the CoherenceProperties of Laser Light........................................................84 Introduction..................................................................................126

Measures to Reduce the Coherence Length of General Remarks on Choice of Excitation LightLaser Light.............................................................................. 84 Sources....................................................................................126

Heat Removal..............................................................................84 Scrambling and Filtering the Light ..........................................131Other Installation Requirements ................................................ 85 Types of Sources and Their Features ......................................132Attenuation of Laser Beams.......................................................85 Structure......................................................................................132

Stabilization of Intensity, Wavelength, and Beam Wavelength................................................................................. 135Position in Lasers......................................................................85 Stability in Time and Wavelength ............................................ 136Sources of Noise in Lasers .........................................................85 Radiance......................................................................................137

Spatial Beam Characteristics......................................................89 Control.........................................................................................138Laser Requirements for Biological Confocal Laser Measuring What Comes Through the

Scanning Microscopy-Related Techniques .........................89 Illumination System................................................................139Optical Tweezers......................................................................... 89 The Bare Minimum....................................................................139Total Internal Reflection Microscopy........................................89 Types of Confocal Microscopes That Can UseConfocal Raman Confocal Laser Scanning Microscopy Non-Laser Light Sources ........................................................141

for Chemical Imaging .............................................................90 Tandem Scanning: Basic Description ........................ .............141Non-Linear Confocal Microscopy.............................................90 Single-Sided Disk Scanning: Basic Description.....................141Nanosurgery and Microdissection ............................................. 90 Exposure Time and Source Brightness ...................................141

Types of Lasers..............................................................................90 Future Trends................................................................................143Continuous Wave Lasers ............................................................ 90

Gas Lasers................................................................................... 90Dye Lasers.................................................................................103 CHAPTER 7: OBJECTIVE LENSES FORSolid-State Lasers..................................................................... 103 CONFOCAL MICROSCOPYThin Disk Lasers .......................................................................109

Pulsed Lasers...............................................................................110 H. Ernst Keller

Classification of Pulsed Laser Systems ................................... 111 Introduction..................................................................................145Nitrogen Lasers.........................................................................112 Aberrations of Refractive Systems..........................................146Excimcr Lasers..........................................................................112 Defocusing..................................................................................146......................Metal Vapor Lasers...................................................................112 Monochromatic Aberrations......................................................147Dye Lasers .................................................................................112 Chromatic Aberrations...............................................................152

Finite Versus Infinity Optics................................................156 Overview....................................................................................207Working Distance................................................................157 Telecentricity............................................................................. 207Optical Materials................................................................. 158 The Scanning System................................................................208Anti-Reflection Coatings .....................................................158 The Back-Focal Planes ............................................................. 210

Transmission of Microscope Objectives ................................. 158 Practical Requirements....................................................... 210Conclusion............................................................................160 Diffraction Limit ....................................................................... 210

Geometric Distortion.................................................................211CHAPTER 8: THE CONTRAST FORMATION Evaluation of the Illumination and DetectionIN OPTICAL MICROSCOPY Systems.............................................................................211

Ping Chin Cheng Influence of Optical Elements ..................................................211Errors..........................................................................................211

Introduction......................................................................... 162 Evaluation of Optical Arrangements ....................................... 212Sources of Contrast.............................................................163 Evaluation of Scanner Arrangements ...................................... 213

Absorption Contrast.................................................................. 163 Scanners..................................................................................... 215Scattering and Reflection Contrast ...........................................167 Attachment to Microscopes......................................................217Phase Contrast ........................................................................... 171 Merit Functions ......................................................................... 217Fluorescence Contrast...............................................................172 Multi-Fluorescence..............................................................217Contrast Related to Excitation Wavelength Special Setups ......................................................................218

Change....................................................................................173 Setups for Fluorescence Recovery AfterNegative Contrast......................................................................173 Photobleaching Experiments ................................................218Special Concerns in Ultraviolet and Near-Infrared Setups for Fluorescence Resonance Energy Transfer

Range Confocal Microscopy ................................................174 Experiments...........................................................................218Total Internal Reflection Contrast ............................................177 Setups for the Integration of Optical Tweezers ......................218Harmonic Generation Contrast ................................................ 179 Setups for the Integration of Laser Cutters............................ 218

Geometric Contrast ............................................................ 180 Setups for the Observation of Living Specimens ...................219z-Contrast in Confocal Microscopy ......................................... 180 Miniaturization and Computer Control.............................219Total Internal Refraction Fluorescence Contrast ....................180 Thermal Stability................................................................. 219Fluorescence Resonant Energy Transfer ................................. 184 Vibration Isolation...............................................................219Fluorescence Recovery After Photobleaching Conclusions and Future Prospects.....................................219

(FRAP and FLIP) .................................................................. 187Structural Contrast.............................................................. 188

Harmonic Generation Contrast .................................................188 CHAPTER 10: DISK-SCANNING CONFOCALBirefringence Contrast .............................................................. 188 MICROSCOPY

Derived Contrast (Synthetic Contrast) ...............................188 Derek Toomre and James B. ParleyRatiometric ................................................................................. 189Deconvolution............................................................................189 Introduction ......................................................................... 221Movement Contrast (Subtraction of Previous Background.......................................................................... 221

I mage).....................................................................................190 Living Cell Imaging: Probing the Future ................................221Spectral Unmixing and Color Reassignment .......................... 190 A Need for Speed and Less Photobleaching.......................... 222Effects of the Specimen: Spherical Aberration and Advantages and Limitations of Confocal

Optical Heterogeneity........................................................... 192 Laser-Scanning Microscopes................................................222Mounting Medium Selection....................................................198 Other Imaging and Deconvolution .......................................... 223

Artificial Contrast ................................................................ 201 Confocal Disk-Scanning Microscopy................................. 223Contrast Resulting from Instrument Vibration and Nipkow Disk - An Innovation............................................... 223

Ambient Lighting..................................................................201 A Renaissance -Advantages of Disk-Scanning

Contrast Resulting from Interference of Cover Confocal Imaging.......................................................... 223_

Glass Surfaces .......................................................................201 Disadvantages............................................................................ 224

Background Level and Ghost Images from the Critical Parameters in Pinhole and Slit Disks ....................224

Transmission Illuminator..................................................... 201 Fill Factor and Spacing Interval F...........................................224

Contrast Resulting from Differences in Lateral Resolution ..................................................................... 225

Photobleaching Dynamics....................................................202 Pinhole/Slit Size........................................................................ 225

Effect of Spectral Leakage and Signal Imbalance Axial Resolution........................................................................225

Between Different Channels ................................................203 Types of Disk-Scanning Confocals .....................................228

New Contrasts: Fluorescence Lifetime and Coherent General Considerations.............................................................228

Antistokes Raman Spectroscopy....................................204 Disk Scanners for Backscattered Light Imaging....................228

Summary..............................................................................204 CARV. DSU. and Other Disk-Scanning ConfocalMicroscopes...........................................................................779

CHAPTER 9: THE INTERMEDIATE OPTICAL The Yokogawa Microlens - An IlluminatingSYSTEM OF LASER-SCANNING CONFOCAL Approach................................................................................2 31MICROSCOPES New Fast Slit Scanner - Zeiss LS,M510 LIVE....................231

New Detectors - A Critical Component ......................... 23 2

Ernst H.K. Stelzer I mage Intensifiers 232

Introduction.........................................................................207 On-Chip Electron Multiplying Charge-Coupled

Design Principles.................................................................207 Device.....................................................................................233

Electron Multiplication Charge-Coupled Devices and Experimental Considerations .............................................. 265Dist. Scanners 234 Pattern Generation ......................................................................266

Applications and Examples of Confocal Computing Optical Sections fromDisk-Scanning Microscopes..............................................235 Structured-Illumination Data..........................................268Comparison with Epi-Fluorescence Imaging .......................... 235 Resolution Improvement by StructuredFast 3D/4D Imaging.................................................................... 235 Illumination................................................................................270Blazingly Fast Confocal Imaging ..............................................235 Nonlinear Structured Illumination ............................................276

Future Developments? .......................................................... 236 Summary 276..................................Summary................................................................................. 237

CHAPTER 11: MEASURING THE REAL POINT SPREAD CHAPTER 14: VISUALIZATION SYSTEMS FORFUNCTION OF HIGH NUMERICAL APERTURE MULTI-DIMENSIONAL MICROSCOPY IMAGESMICROSCOPE OBJECTIVE LENSES N.S. White

Riinas Juskaitis Introduction 280................................Introduction............................................................................239 Definitions................................................................................... 280

Measuring Point Spread Function 240 What Is the Microscopist Trying to Achieve?........................280

Fiber-Optic Interferometer......................................................... 240 Criteria for Choosing a Visualization System........................ 281

Point Spread Function Measurements ...................................... 241 Why Do We Want to Visualize Multi-Dimensional

Chromatic Aberrations ..........................................................242 Laser-Scanning Microscopy Data? .................................. 281

Apparatus ...................................................................................... 243 Data and Dimensional Reduction.............................................281

Axial Shift .....................................................................................243 Objective or Subjective Visualization? ....................................281

Pupil Function ........................................................................ 245 Prefiltering................................................................................... 281

Phase-Shifting Interferometr 245 Identifying Unknown Structures ...............................................281

Zernike Polynomial Fit...............................................................245 Highlighting Previously Elucidated Structures .......................284

Restoration of a 3D Point Spread Function .............................247 Visualization for Multi Dimensional

enure 248 Measurements......................................................................... 284Empty APMiscellanea.............................................................................248 What Confocal Laser Scanning Microscopy Images............. . .

Temp erature Variations 248 Can the Visualization System Handle? ...........................286.......................Polarization Effects......................................................................249 Image Data: How Are Image Values Represented

250 in the Program? 286Apodization.........................................................................Conclusion 250 What Dimensions Can the Images and

Views Have? ........................................................................... 286Standard File Formats for Calibration and

CHAPTER 12: PHOTON DETECTORS FOR Interpretation 288CON FOCAL MICROSCOPY How Will the System Generate the ReconstructedJonathan Art Views?.................................................................................290

Assessing the Four Basic Steps in the GenerationIntroduction.................................................................................... 251 of Reconstructed Views 290.....................The Quantal Nature of Light................................................251 Loading the Image Subregion ...................................................290Interaction of Photons with Materials................................252 Choosing a View: The 5D Image Display Space................... 291

Thermal Effects ............................................................................252 Mapping the Image Space into the Display Space .................294Direct Effects................................................................................252 How Do 3D Visualizations Retain thePhotoconductivity ........................................................................ 252 Information?.........................................................................296Photovoltaic ..................................................................................252 Mapping the Data Values into the Display ............................. 300Photoemissive...............................................................................254 How Can Intensities Be Used to RetainComparison of Detectors ............................................................255 Information?.........................................................................304

Noise Internal to Detectors ................................................. 256 Hidden-Object Removal............................................................ 304Noise in Internal Detectors ........................................................ 256 Adding Realism to the View .....................................................306Noise in Photoemissive Devices ...............................................256 How Can I Make Measurements Using theStatistics of Photon Flux and Detectors...................................257 Reconstructed Views? ............................................................. 312Representing the Pixel Value .....................................................258 Conclusion 312

Conversion Techniques .........................................................259

Assessment of Devices ..........................................................260

Point Detection Assessment and Optimization .......................260 CHAPTER 15: AUTOMATED THREE-Field Detection Assessment and Optimization ........................261 DIMENSIONAL IMAGE ANALYSIS METHODS

Detectors Present and Future..............................................262 FOR CONFOCAL MICROSCOPY

CHAPTER 13: STRUCTURED ILLUMINATION Badrinath Rovsam, Gang Lin, Muhammad-Amri Abdul-Karim,

METHODSOmar Al-Kofahi, Khalid Al-Kofahi, William Shalt],Donald H. Szarorrsk, and James N. Turner

Rainer Heint,nuamIntroduction.......................................................................... 316

Introduction...........................................................................265 Types of Automated Image Analysis Studies .....................318

Common Types of Biological Image Objects......................319 Oxygen Sensor..........................................................................347Specimen Preparation and Image Preprocessing cAMP Indicators................................................................ 347

Methods........................................................................... 319 Fatty Acid Indicator ............................................................347Data Collection Guidelines for Image Analysis Genetically Expressed Intracellular Fluorescent

Purposes .........................................................................319 Indicators..........................................................................345Image Preprocessing Methods............................................ 320 Green Fluorescent Protein ...................................................348

General Segmentation Methods Applicable to Ligand-Binding Modules....................................................345Confocal Data..................................................................321 Ion Indicators.....................................................................348Bottom-Up Segmentation Methods .....................................321 Future Developments..........................................................348Top-Down Segmentation Methods ......................................322Hybrid Segmentation Methods Combining Bottom-Up

and Top-Down Processing 322 CHAPTER 17: PRACTICAL CONSIDERATIONS INExample Illustrating Blob Segmentation.............................. 322 THE SELECTION AND APPLICATION OF

Model-Based Object Merging................................................. 323 FLUORESCENT PROBESExample Illustrating Segmentation of Tube-Like

Objects............................................................................. 324 lain D. JohnsonSkeletonization Methods .....................................................3224 Introduction 353Vectorization Methods.................... 374 Selection Criteria for Dyes and Probes......................... 353

Example Combining Tube and Blob Organic Dyes..................... .................. 353Segmentation 328 Fluorescent Proteins: Green Fluorescent Protein andRegistration and Montage Synthesis Methods................... 328

Methods for Quantitative Morphometry.......................... 331Phvcobiliproteins............................................................ 356

Methods for Validating the Segmentation andQuantum Dots 357

Making Corrections ...............................................................333 Multi Photon Excitation 357

Analysis of Morphometric Data.................................................... Introducing the Probe to the Specimen ............................ 358.................334 Loading methods ................................................................358Discussion, Conclusion, and Future Directions ..................335 Target Abundance and Autofluorescence

Considerations................................................................ 360

CHAPTER 16: FLUOROPHORES FOR CONFOCAL Interactions of Probes and Specimens................................. 361

MICROSCOPY: PHOTOPHYSICS ANDLocalization and Metabolism.............................................. 361Perturbation and Cytotoxicity ............................................. 362

PHOTOCHEMISTRY Under the Microscope........................................................ 362Roger Y. Tsien, Lauren Ernst, and Alan Waggoner Photobleaching.....................................................................................362

IntroductionPhototoxicity......................................................................363

I .....................................................................338 Summary ,64Photophysical Problems Related to High Intensity

Excitation..........................................................................338Singlet State Saturation...................................................... 338Triplet State Saturation ............................................................ 339 CHAPTER 18: GUIDING PRINCIPLES OFContaminating Background Signals.................................... 339 SPECIMEN PRESERVATION FOR CONFOCALWhat Is the Optimal Intensity?................................................340 FLUORESCENCE MICROSCOPY

Photodestruction of Fluorophores and Biological Robert ßurallun, Sada(.Snhrah, and Carrie PhillipsSpecimens........................................................................ 340Dependency on Intensity or Its Time Integral?....................34)) Introduction .................................................................................368

Strategies for Signal Optimization in the Face of Characteristics of Fixatives ......................................................368Photobleaching................................................................341 Glutaraldehyde................................................................... 369Light Collection Efficiency.................................................341 Formaldehyde.....................................................................369Spatial Resolution.....................................................................341 Fixation Staining and Mounting Methods.........................370Protective Agents ................................................................341 Glutaraldehyde Fixation......................................................37(1Fluorophore Concentration................................................. 342 pH Shift/Formaldehyde Fixation .........................................370Choice of Fluorophore........................................................342 l mmunofluorescencc Stainin g...................................................................................371

Fluorescent Labels for Antibodies, Other Proteins, Mounting the Specimen...........................................................371and DNA Probes.............................................................. 342 Critical Evaluation of Light Microscopy Fixation andFluorescent Organic Dyes...................................................342 Mounting Methods..........................................................371

Phycobiliproteins................................................................343 Use of the Cell Height to ENaluate theDNA Probes .......................................................................343 Fixation Method............................................................. 372Luminescent Nanocrystals .................................................. 343 Use of Cell Height to Es aluate MountingFluorescent Lanthanide Chelates .........................................345 Media.............................................................................373

Fluorescent Indicators for Dynamic Intracellular 'Veil-Defined Structures Can Be Used to EsaluateParameters ........................................................................346 Fixation Methods............................................................373Membrane Potentials.......................................................... 346 Comparison of In lino Labeled Cell Organelles withIon Concentrations..............................................................346 lmmunolahelcd Cell Organelles......................................374

p H Indicators ......................................................................346 General Notes......................................................................374

Ca' Indicators.................................................................... 346 Labeling Samples with Two or More Probes.........................375

Triple Labeling........................................................................... 375 CHAPTER 20: ABBERATIONS IN CONFOCALPreparation of Tissue Specimens ..............................................376 AND MULTI-PHOTON FLUORESCENCELabeling Thick Sections............................................................ 376 MICROSCOPY INDUCED BY REFRACTIVERefractive Index Mismatch....................................................... 377 INDEX MISMATCHScreening Antibodies on Glutaraldehyde-Fixed

377Alexander Egner and Stefan W. Hell

Specimens........................................................................Microwave Fixation ................................................................... 377 Introduction.................................................................................. 404

Conclusion.....................................................................................378 The Situation.................................................................................404Theory.............................................................................................404Results of Theoretical Calculations........................................ 407Experiments...................................................................................409

CHAPTER 19: CONFOCAL MICROSCOPY Other Considerations.................................................................410OF LIVING CELLS Dry Objectives ............................................................................410

Michael E. Daher. Erik Manders, David R. Soll,Refractive Index, Wavelength, and Temperature ....................411

and Mark Tero.ccikiSpherical Aberration Correction............................................... 411

Conclusion.....................................................................................412Introduction.................................................................................. 381 Consequences..............................................................................412Overview of Living-Cell Confocal I maging Practical Strategies to Reduce Refractive Index

Techniques.................................................................................382 Mismatch.................................................................................412Time-Lapse Fluorescence Imaging.......................................... 382Multi-Channel Time-Lapse Fluorescence Imaging ................ 382 CHAPTER 21: INTERACTION OF LIGHT WITHSpectral Imaging and Linear Unmixing ..................................382Fluorescence Recovery After Photobleaching ........................ 382 BOTANICAL SPECIMENS

Fluorescence Loss in Photobleaching......................................382 Ping-Chin ChengFluorescence Resonance Energy Transfer ...............................382Fluorescence Lifetime Imaging................................................382 Introduction.................................................................................. 414

383Light Attenuation in Plant Tissue.............................................414

Fluorescence Correlation Spectroscopy ...........................Fluorescence Speckle Microscopy........................................... 383 Linear Absorption.......................................................................414

OpticPhoto-Uncag mg/Photoactivation 383 Nonlinear Absorption.................................................................416

al Tweezers/Laser Trapping 383 Scattering.................................................................................... 417

Physiological Fluorescence Imaging 383 Refractive Index Heterogeneity ...............................................418

Combining Fluorescence and Other Imaging Birefringent Structures in Plant Cells .....................................420

Modalities................................................................................383 Fluorescence Properties of Plants...........................................421

General Considerations for Confocal Microscopy Changes in Emission Spectra Depending on

of Living Cells...........................................................................386 One- Versus Two-Photon Excitation ....................................421

Maintenance of Living Cells and Tissue Microspectroscopy 421Light-Specimen Interaction (FluorescencePreparations 387

Emission)................................................................................ 425Fluorescent Probes.....................................................................387Minimizing Photodynamic Damage........................................ 389 Harmonic Generation Properties 428

The Effect of Fixation on the Optical PropertiesThe Online Confocal Community ..........................................390of Plants..................................................................................... 428A Convenient Test Specimen ....................................................390

Specific Example I: Visualizing Chromatin Living Plant Cells ..........................................................................429

Dynamics Using Very Low Light Levels .............................390 Callus, Suspension Culture Cells and

Phototoxicity 390 Protoplasts .............................................................................. 429

Reduction of Phototoxicity.................. 391 Meristem 430

I mproving Image Quality in Low-Dose Stem and Root 430

Microscopy............................................................................. 391 Microspores and Pollen Grains................................................ 431

Low-Dose Imaging Conclusion ................................................391 Cuticles, Hairs, and Waxes 434

Specific Example II: Multi-Dimensional Imaging Storage Structures...................................................................... 435

of Microglial Cell Behaviors in Live Rodent Mineral Deposits ........................................................................ 436

Brain Slices 392 Primary and Secondary Cell Walls .......................................... 438...............................Preparation of Central Nervous System Tissue Fungi............................................................................................438

Slices.......................................................................................393 Conclusion.....................................................................................439

Fluorescent Staining..................................................................393Maintaining Tissue Health on the Microscope CHAPTER 22: SIGNAL-TO-NOISE RATIO IN

Stage 393 CONFOCAL MICROSCOPESI maging Methods....................................................................... 394I maging Deep Within Tissue .................................................... 391 Cohn J.R. Sheppard. Xiaosong Gan, Min Go,

Keeping Cells in Focus .............................................................395 and Maitreyee Roy

Handling the Data......................................................................395 Introduction..................................................................................442Results ........................................................................................ 396 Sources of Noise..........................................................................442Conclusion..................................................................................396 Shot Noise and Quantum Efficiency........................................442

Future Directions........................................................................ 398 Background Noise......................................................................443

Signal Level in Confocal Microscopes ............................... 444 More Examples....................................................................480Signal-to-Noise Ratio for Confocal Blind Deconvolution and Spherical Aberration................... 480

Microscopes.....................................................................445 Widefield Fluorescence Simulation .....................................481Q,, NI, and Stain Level ......................................................445 Spinning-Disk Confocal..................................................... 481N2 and Detectability.......................................................... 446 Two Photon........................................................................481Multi-Photon Fluorescence Microscopy ..............................447 Speed....................................................................................482

Designs of Confocal Microscopes ......................................447 Future Directions .................................................................483Sampling .............................................................................. 448 Summary of Main Points .................................................... 483Comparative Performance of Fluorescence

Microscopes.....................................................................448Bleaching-Limited Performance......................................... 448 CHAPTER 25: I MAGE ENHANCEMENT BYSaturation-Limited Performance......................................... 450 DECONVOLUTIONEffects of Scanning Speed........................................................450

Mark B. Connell, Angus McMorland, am/ Christian Seel/er3D Imaging........................................................................451Summary..............................................................................451 Introduction.........................................................................488

Background ..........................................................................488

CHAPTER 23: COMPARISON OF Image Formation......................................................................... 489

WIDEFIELD/DECONVOLUTION ANDForwards: Convolution and the I maging System................490

CONFOCAL MICROSCOPY FOR THREE- Properties of the Point Spread Function...........................492

DIMENSIONAL IMAGING Quantifying the Point Spread Function.............................492The Missing Cone Problem .................................................494

Peter J. Shaw Noise.................................................................................... 495

Introduction 453Deconvolution Algorithms ..................................................495................................

The Point Spread Function: Imaging as aNearest-Neighbor Deconvolution ........................................495

Convo lution....................................................................................Wiener Filtering ................................................................. 496C 453

Nonlinear Constrained Iterative DeconvolutionLimits to Linearity and Shift Invariance................................. 457

Deconvolution 457 Algorithms..................................................................... 496

Practical Differences ........................................................... 458 Comparison of Methods..................................................... 497

Temporal Resolution.......................................................... 458Combination of Charged-Coupled Device and Confocal CHAPTER 26: FIBER-OPTICS IN SCANNING

Imaging ..........................................................................458 OPTICAL MICROSCOPYIntegration of Fluorescence Intensity.................................. 459

Resolution, Sensitivity, and Noise...................................... 459 Peter Delaney and Martin Harri.sFluorescence Excitation......................................................459 Introduction " 501Fluorescent Light Detection ................................................459 l"R l' " "' " " " 'Gain Register Charge-Coupled Devices..................................460

Key Fiber Technologies Relevant to ScanningMicroscopy.......................................................................501

Out-of-Focus Light ....................................................................461 Glass Made from Gas and its TransmissionModel Specimens...............................................................461 Properties........................................................................501The Best Solution: Deconvolving Confocal Data ..................461 Step Index and Gradient Index Optical Fibers.......................501

Practical Comparisons ........................................................ 463Conclusion

Modes in Optical Fibers..................................................... 502C .........................................................................466 Evanescent Wave and Polarization Effects inSummary.............................................................................. 467

Optical Fibers 503Polarization-Maintaining Fibers..........................................503

CHAPTER 24: BLIND DECONVOLUTION Fused Biconical Taper Couplers: Fiber-Optic

Timothy J. Holmes, David Biggs, and Asad Ahu-Tarif MicrostructureF FiSplitte......................................................................... 503

bers......................................................... 504Introduction.........................................................................468 Fiber Image Transfer Bundles .............................................504

Purposes of Deconvolution.......................................................468 Key Functions of Fibers in Optical Microscopes................505Advantages and Limitations............................................... 468 Optical Fiber for Delivering Light ...................................... 505

Principles..............................................................................472 Optical Fiber as a Detection Aperture .................................506Data Collection Model ....................................................... 472 Same Fiber for Both Source and Confocal

Maximum Likelihood Estimation ........................................472 Detection........................................................................506Algorithms......................................................................... 472 Fiber Delivery for Nonlinear Microscopy with

Different Approaches ..........................................................475 Femtosecond Lasers ........................................................5073D......................................................................................475 Large Core Fibers as Source or Detection2D Image Filtering.............................................................476 Apertures ........................................................................507

Data Corrections ................................................................. 477 Benchtop Scanning Microscopes Exploiting FiberLight Source and Optics Alignment ...................................477 Components.....................................................................507Newest Developments........................................................478 Miniaturized Scanning Confocal Microscope

Subpixel.....................................................................................478 I maging Heads.................................................................508Polarized Light ...................................................................479 Miniature Confocal Imaging Heads Based onLive Imaging ......................................................................480 Coherent Imaging Bundles ..............................................508

Resolution and Optical Efficiency of Bundles ........................509 Chromophores (Fluorophores and CagedBundle Imagers for In Vi vo Studies in Animals..................... 509 Compounds)............................................................................. 543Scan Heads Based on Single Fibers with Miniature Two-Photon Absorption Cross-Sections.................................. 543

Scanning Mechanisms ...........................................................510 Caged Compounds..................................................................... 544Vibrating the Fiber Tip..............................................................510 Cell Viability During Imaging..............................................544Vibrating the Lens and Fiber ....................................................510 Applications.......................................................................... 545Scanning with Micromirrors.....................................................511 Calcium Imaging........................................................................545Scanning Fiber Confocal Microscopes for In Vivo Uncaging and Photobleaching .................................................. 545

I maging in Animals............................................................... 512 Autofluorescence........................................................................545I mplementations for Clinical Endomicroscopy.................513 Developmental Biology............................................................. 545Summary.............................................................................. 513 In Vivo (Intact Animal) Imaging...............................................545

Outlook.................................................................................545

CHAPTER 27: FLUORESCENCE LIFETIME IMAGING INSCANNING MICROSCOPY CHAPTER 29: MULTIFOCAL MULTI-PHOTONH.C. Gerritsen, A. Draaijer, D.J. van den Heuvel, MICROSCOPYand A.V. Agronskaia

nIJ6rg Bewersdorf Alexander Egner, and Stefan W. Hell

t roduction......................................................................... 516Fluorescence, Lifetime, and Quantum Introduction.................................................................................. 550

Efficiency................................................................................516 Background.................................................................................550Determination of the Optimum Degree ofFluorescence Lifetime Spectroscopy 516

Parallelization.........................................................................550Fluorescence Lifetime Imaging Applications ......................... 516Experimental Realization 551Fluorescence Resonance Energy Transfer ...............................517

Fluorescence Lifetime Imaging Methods ...........................518 A Multi-Focal Multi-Photon Microscopy Setup Using

Introduction 5l8 a Nipkow-Type Microlens Array......................................... 551............................................................................. 5Lifetime Sensing in the Frequency Domain ........................... 518 Resolution

Multiplexing.........Solution ..............................................552

Fluorescence Lifetime Sensing in the Time Time Multiplexing as a to Interfocal

Domain................................................................................... 520 Crosstalk................................................................................. 553

Comparison of Confocal Fluorescence Lifetime Alternative Realizations ............................................................ 554

523 Advanced Variants of Multi-Focal Multi-PhotonImaging Methods............................................................527 Microscopy 555Applications...................................................................

Multi-Labeling and Segmentation ............................................527 Space Multiplexing .................................................................... 555

Ion-Concentration Determination.............................................528 Fluorescence Lifetime Imaging 555

Probes for Fluorescence Lifetime Microscopy.......................530 Second Harmonic Generation Multi-Focal Multi-Photon

Summary.............................................................................. 532 Microscopy............................................................................. 556Multi-Focal Multi-Photon Microscopy-4Pi

Microscopy............................................................................. 556

CHAPTER 28: MULTI-PHOTON MOLECULAR I maging Applications........................................................... 556

EXCITATION IN LASER-SCANNING Limitations............................................................................ 556

MICROSCOPY Current Developments ........................................................558Summary...............................................................................559

Winfried Denk, David W Piston, and Watt W. Webb

Introduction......................................................................... 535Physical Principles of Multi-Photon Excitation CHAPTER 30: 4Pi MICROSCOPY

and Their Implications for Image Formation .................535Jorg Bewersdorf, Alexander Egner, and Stefan W. HellPhysics of Multi-Photon Excitation .........................................535

Optical Pulse Length .................................................................537 Introduction..........................................................................561Excitation Localization ............................................................. 538 Theoretical Background.......................................................562Detection.................................................................................... 538 The Point Spread Function........................................................562Wavelengths...............................................................................538 The Response and the Axial Resolution ...............................563Resolution.................................................................................. 539 The Optical Transfer Function..................................................563Photodamage: Heating and Bleaching .....................................539 Multi-Focal Multi-Photon Microscopy-4Pi

Instrumentation 540 Microscopy........................................................................563Lasers and the Choice of Excitation Wavelengths .................540 Space Invariance of the Point Spread Function ..................... 564Detection.................................................................................... 541 Live Mammalian Cell 4Pi Imaging ......................................... 564Optical Aberrations................................................................... 542 Type C 4Pi Microscopy with the Leica TCS 4P1.................565Pulse Spreading Due to Group Delay Dispersion ..................543 Resolution................................................................................... 567Control of Laser Power ............................................................ 543 Type C 4Pi Imaging in Living Cells ........................................568Resonance and Non-Mechanical Scanning ............................. 543 Summary and Outlook ........................................................568

CHAPTER 31: NANOSCALE RESOLUTION WITH Coherent Anti-Stokes Raman Scattering CorrelationFOCUSED LIGHT: STIMULATED EMISSION Spectroscopy....................................................................602DEPLETION AND OTHER REVERSIBLE SATURABLE Coherent Anti-Stokes Raman Scattering MicroscopyOPTICAL FLUORESCENCE TRANSITIONS Imaging of Biological Samples........................................603MICROSCOPY CONCEPTS Conclusions and Perspectives ............................................ 604

Stefan W. Hell, Katrin L Willig, Marcus Dyba,Stefan Jakobs, Lars Kastrup, and Volker Westphal

The Resolution Issue 571 CHAPTER 34: RELATED METHODS FOR

Breaking the Diffraction Barrier: The Concept of THREE-DIMENSIONAL IMAGING

Reversible Saturable Optical Fluorescence J. Michael Tvszka, Seth W. Ruffins, Jamey P. Weicher!,Transitions........................................................................ 571 Michael J. Paulus, and Scott E. Fraser

Different Approaches of Reversible Saturable Optical Introduction.........................................................................607Fluorescence Transitions Microscopy............................ 573

Stimulated Emission Depletion Microscopy ..................... 574Surface Imaging Microscopy and Episcopic

Challenges and Outlook.....................................................577Fluorescence Image Capture ..........................................607

Optical Coherence Tomography ........................................609Optical Projection Tomography ......................................... 610Light Sheet Microscopy ...................................................... 613

CHAPTER 32: MASS STORAGE, DISPLAY, Optical Setup............................................................................. 613AND HARD COPY Micro-Computerized Tomography Imaging ......................614

Guy CoxOperating Principle ................................................................... 614Contrast and Dose .....................................................................614

Introduction.........................................................................580 Computed Tomography Scanning Systems ............................ 615Mass Storage........................................................................580 Magnetic Resonance Microscopy...................................... 618

Data Compression .....................................................................580 Basic Principles of Nuclear MagneticRemovable Storage Media ....................................................... 585 Resonance..............................................................................618Random-Access Devices.......................................................... 586 Magnetic Resonance Image Formation ...................................619Solid State Devices................................................................... 588 Magnetic Resonance Microscopy Hardware.......................... 622

Display..................................................................................588 Strengths and Limitations of Magnetic ResonanceMonitors .....................................................................................588 Microscopy............................................................................622Liquid Crystal Displays............................................................589 Image Contrast in Magnetic Resonance Microscopy............622Data Projectors .......................................................................... 590 Magnetic Resonance Microscopy Applications ......................623

Hard Copy........................................................................... 590 Future Development of Magnetic ResonancePhotographic Systems...............................................................590 Microscopy............................................................................ 624Digital Printers.......................................................................... 591 Conclusion........................................................................... 624

Conclusion........................................................................... 593Summary.............................................................................. 593

Bulk Storage.............................................................................. 593 CHAPTER 35: TUTORIAL ON PRACTICALDisplay....................................................................................... 594 CONFOCAL MICROSCOPY AND USE OF THEHard Copy..................................................................................594 CONFOCAL TEST SPECIMEN

Victoria Centonze and James B. Pawlev

CHAPTER 33: COHERENT ANTI-STOKES Introduction.........................................................................627RAMAN SCATTERING MICROSCOPY Getting Started...........................................................................627

X. Sunney Xie, Ji-Xin Cheng, and Eric PotnzaBleaching -The Only Thing That Really

Matters....................................................................................627Introduction.........................................................................595 Getting a Good Confocal Image ........................................629Unique Features of Coherent Anti-Stokes Raman Simultaneous Detection of Backscattered Light

Scattering Under the Tight-Focusing Condition........... 596 and Fluorescence...................................................................631Forward and Backward Detected Coherent New Controls...................................................................... 631

Anti-Stokes Raman Scattering........................................597 Photon Efficiency......................................................................631Optimal Laser Sources for Coherent Anti-Stokes Pinhole Size ...............................................................................63 1

Raman Scattering Microscopy........................................599 Stray Light .................................................................................632Suppression of the Non-Resonant Background................600 Is the Back-Focal Plane Filled? ...............................................633

Use of Picosecond Instead of Femtosecond Pulses...............600 Pinhole Summary......................................................................633Epi-Detection.............................................................................600 Statistical Considerations in ConfocalPolarization-Sensitive Detection ..............................................600 Microscopy............................................................................ 633Time-Resolved Coherent Anti-Stokes Raman Scattering The Importance of Pixel Size .................................................. 634

Detection................................................................................600 Measuring Pixel Size ................................................................635Phase Control of Excitation Pulses ......................................... 600 Over-Sampling and Under-Sampling......................................635

Multiplex Coherent Anti-Stokes Raman Scattering Nyquist Reconstruction and Deconvolution...........................635Microspectroscopy..........................................................602 Pixel Size Summary..................................................................636

Using a Test Specimen .........................................................636 Absorbers and Targets in Biological Specimens .................... 682

Why Use a Test Specimen?......................................................636 Laser Exposure Parameters .......................................................682

Description of the Test Specimen ............................................636 Evidence for Near Infrared-Induced Reactive Oxygen

Using the Test Specimen ...........................................................637 Species Formation..................................................................683

The Diatom: A Natural 3D Test Specimen ............................. 638 Evidence for Near Infrared-Induced DNA Strand

Reasons for Poor Performance ...........................................640 Breaks......................................................................................684

Sampling Problems....................................................................640 Photodynamic-Induced Effects.................................................684

Optical Problems ........................................................................640 Photothermal Damage.........................................................685

I maging Depth............................................................................643 Damage by Optical Breakdown..........................................685

Singlet-State Saturation .............................................................643 Modifications of Ultrastructure...........................................685

Which 3D Method Is Best?................................................. 644 Influence of Ultrashort Near Infrared Pulses onOptimal 3D Light Microscopy Summary ................................646 Reproductive Behavior.......................................................... 686

Things to Remember About Deconvolution........................... 646 Nanosurgery................................................................................. 686

Decision Time............................................................................ 646 Conclusion............................................................................687

Multi-Photon Versus Single-Photon Excitation ................. 646Widefield Versus Beam Scanning ............................................ 647

Summary...............................................................................647 CHAPTER 39: PHOTOBLEACHING

Alberto Diaspro, Giuseppe Chirico, Cesare Usai,CHAPTER 36: PRACTICAL CONFOCAL MICROSCOPY Paola Ramoino, and Jurek Dobrucki

Alan R. Hibbs, Glen MacDonald, and Karl Garsha Introduction..................................................................................690

The Art of Imaging by Confocal Microscopy .................... 650 Photobleaching.................................................................... 691

Balancing Multiple Parameters 650 Photobleaching Mechanisms .....................................................691

Monitoring Instrument Performance................................. 650 Reducing Photobleaching ..........................................................693

Illumination Source.............................................................650 Photobleaching at the Single-Molecule Level ...................696Photobleaching of Single Molecules....................................... 697Scan Raster and Focus Positioning 651Photobleaching and Photocycling of SingleOptical Performance and Objective Lenses ............................652

Signal Detection 660 Fluorescent Proteins.............................................................. 698

Optimizing Multi-Labeling Applications ............................663 Bleaching and Autofluorescence 698

Control Samples Establish the Limits .....................................663 Other Fluorescent Proteins........................................................698

Separation of Fluorescence into Spectral Regions .................664 Conclusion.....................................................................................699

Sequential Channel Collection to MinimizeBleed-Through .......................................................................664

Spectral Unmixing 664 CHAPTER 40: NONLINEAR (HARMONICColocalization 667 GENERATION) OPTICAL MICROSCOPY..............................

I mage Collection for Colocalization ........................................667 Ping-Chin Cheng and C.K. SunQuantifying Colocalization .......................................................668

IntroductionSpatial Deconvolution in Colocalization Studies...................668 • • 703

Discussion 670 Harmonic Generation..........................................................704.................................Second Harmonic Generation...................................................704Third Harmonic Generation......................................................705CHAPTER 37: SELECTIVE PLANE ILLUMINATION Multi-Photon Absorption and Fluorescence ........................... 705MICROSCOPY

Light Sources and Detectors for Second HarmonicJan Huisken, Jim Swage!: Steffen Lindek, Generation and Third Harmonic Generationand Ernst H .K. Stelzer I maging............................................................................. 706

Nonlinear Optical Microscopy Setup.................................708Introduction ... . 672 Optically Active Biological Structures................................710Combining LightSheet

Sheet Illumination andOptically Active Structures in Plants.......................................710Orthogonal Detection.....................................................672 Optically Active Structures in AnimalSelective Plane Illumination Microscopy Setup ................673 Tissues 714.................................Lateral Resolution................................................................674 Polarization Dependence of Second HarmonicLight Sheet Thickness and Axial Resolution......................674 Generation 717Applications...................................................................................................................................................................................................................................... 675 Summary 719Processing Selective Plane Illumination Microscopy ...............................................................................' ................ ' ..'

I mages/Multi-View Reconstruction................................675Summary..............................................................................678 CHAPTER 41: IMAGING BRAIN SLICES

CHAPTER 38: CELL DAMAGE DURING Avuntu Tashiro, Gloster Aaron, DmitrivAronnr,

MULTI-PHOTON MICROSCOPY Rosa Cossart, Daniella Dumitriu, Vivian Fenstermaker,Jesse Goldberg, Farid Harnzei-Sichani, Yuji Ikegava,

Karsten KrinigSvla Konut; Jason MacLean, Boaz Newt,

Introduction 680 Volodvrnvr Nikolenko, Carlos Portera-Cailliau,Photochemical Damage in Multi-Photon and Rafael Yuste

Microscopes .....................................................................682 Introduction..........................................................................722

Making Brain Slices.............................................................722 The Quest for Better Resolution: Aberration andAcute Slices....................................................................... 722 the Challenge of Imaging Thick Embryos ....................... 747Cultured Slices ...................................................................724 Embryos Are Highly Scattering and Refractile

Labeling Cells ...................................................................... 724 Specimens...................................................................... 747Biolistic Transfection ......................................................... 724 Imaging Embryos Involves Inherent Trade-Offs ....................747Genetic Manipulation with Dominant-Negative and Common Themes in Living Embryo Imaging Have

Constitutively Active Mutants.........................................725 System-Specific Solutions...............................................748Diolistics and Calistics....................................................... 726 Dealing with Depth: Strategies for ImagingDye Injection with Whole-Cell Patch Clamp......................726 Thick Specimens.....................................................................748Slice Loading and "Painting" with Acetoxymethyl Avoiding the Thickness Dilemma: Going Small..................748

Ester Indicators...................................................................... 726 Grazing the Surface: Superficial Optical SectionsGreen Fluorescent Protein Transgenic Mice...........................727 Are Often Sufficient..............................................................748

I maging Slices......................................................................727 Up from the Deep: Explants Can Reduce theTwo-Photon Imaging of Slices............................................727 Thickness of Specimens Dramatically.............................748Slice Chamber Protocol ......................................................727 Multi-Photon Microscopy Can Penetrate MoreChoice of Objectives ..........................................................727 Deeply into Specimens................................................... 749Beam Collimation and Pulse Broadening........................... 728 Selective Plane Illumination Can Provide OpticalImage Production, Resolution, and z-Sectioning ....................729 Sectioning in Very Thick Specimens...................................751Choice of Indicators for Two-Photon Imaging of Deconvolution and Other Post-Acquisition

Calcium..........................................................................729 Processing ...................................................................... 751Photodamage......................................................................729 Striving for Speed: Strategies for ReducingSecond Harmonic Imaging................................................. 729 Specimen Exposure 753Silicon-Intensified Target Camera Imaging ............................ 730 Simple Solutions: Reducing Image Dimensions.

Morphological Processing and Analysis................................ 730 Increasing Slice Spacing, and Scan Speed.......................753Biocytin Protocol .......................................................................730 Disk-Scanning Confocal Microscopy AllowsAnatomy with a Two-Photon/Neurolucida High-Speed Acquisition ..................................................754

System.....................................................................................731 Additional Hardware Improvements Can IncreaseCorrelated Electron Microscopy......................................... 731 Acquisition Speed ...........................................................754Morphological Classification of Neurons Using Localizing Label: Strategies for Increasing

Cluster Analysis............................................................. 731 Effective Contrast in Thick Specimens...........................755Image Processing .................................................................732 Addition of Labeled Proteins to Embryos ........................... 756

Compensation for the Drift and the Vibration of Expressing Green Fluorescent Protein and mRFPthe Slices........................................................................732 Constructs in Embryos Allows Dynamic Analysis of

Alignment Based on the Overlap Between Embryos at Multiple Wavelengths.................................. 756Images............................................................................732 Using Selective Labeling to Reduce the Number

Alignment Based on the Center of Mass ................................ 732 of Labeled Structures............................................................ 757Online Cell Detection of Neurons ........................................... 733 Bulk Vital Labeling Can Enhance Contrast ............................760Image De-Noising Using Wavelets .....................................734 Seeing in Space: Strategies for 4D Visualization...............761

Summary....................................................................................... 734 Depicting Embryos in Time and Space: 2D + TimeVersus 3D + Time...........................................................762

Other Uses for Confocal and Multi-PhotonCHAPTER 42: FLUORESCENT ION Microscopy in Imaging and ManipulatingMEASUREMENT Embryos............................................................................764

Multi-Photon-Based Ablation.............................................764Mark B. Cannel/ and Stephen H. Cody Fluorescence Resonance Energy Transfer...........................764Introduction.........................................................................736 Conclusions: A Bright Future for 3D Imaging of

The Limiting Case..............................................................736 Living Embryos.................................................................766Choice of Indicator.............................................................737Introducing the Indicators into Cells.................................738Care of Fluorescent Probes................................................ 739 CHAPTER 44: IMAGING PLANT CELLSInterpretation of Measurements ........................................740

Nuno Moreno, Susan Boagourd. Jinn Haseloft:Kinetics .................................................................................741 and Jose A. FeijdCalibration............................................................................742Conclusion........................................................................... 745 Introduction.........................................................................769

The Ever Present Problem of Autofluorescence..................770Single-Photon Confocal Microscopy .................................772

CHAPTER 43: CONFOCAL AND MULTI-PHOTON Staining Plant Tissues................................................................774I MAGING OF LIVING EMBRYOS Clearing Intact Plant Material .............................................774

3D Reconstruction ..............................................................775Jeff Hardin 3D Segmentation 776Introduction.........................................................................746 Two-Photon Excitation: Are Two BetterInto the Depths: Embryos Are Thick, Refractile, Than One?.................................................................................778

and Susceptible to Photodamage.......................................746 I mproved Signal-to-Noise Ratio and DynamicImaging Embryos Often Requires "4D" Imaging..................746 Range...................................................................................... 778

I maging Thick/Opaque Specimens ...........................................779 Transfection Reagents ................................................................803Fading, Vital Imaging, and Cell Viability ................................779 Microinjection.............................................................................803Two-Photon Imaging of Plant Cells and Future Perspectives: 3D Microscopy, Biological

Organelles ................................................................................782 Complexity, and In Vivo Molecular Imaging..................804Two-Photon Excitation Imaging of Green Fluorescent In Vivo Molecular Imaging...................................................806

Protein ......................................................................................782Dynamic Imaging..................................................................783Deconvolution 784 CHAPTER 46: AUTOMATED CONFOCAL

Conclusion.............................................................................785 I MAGING AND HIGH-CONTENT SCREENING FORCYTOMICS

Muria A. DeBernardi, Stephen M. Hewitt,CHAPTER 45: PRACTICAL FLUORESCENCE and Andres KrieteRESONANCE ENERGY TRANSFER ORMOLECULAR NANOBIOSCOPY OF Introduction..........................................................................809

LIVING CELLS Platforms Used for Automated ConfocalI maging..............................................................................810

Irma Majoul, Yiwei Jia, and Rainer Duden Types of Assays ..................................................................... 811

Introduction 788 3D Cell Microarray Assays ...................................................815................................How to Make a Good Science..................................................788 Data Management and Image Informatics ........................816

Beauty, Functionality, Cell Cycle, and Conclusion.....................................................................................817

Living-Cell Imaging...............................................................790Fluorescence Resonance Energy Transfer Theory .............790 CHAPTER 47: AUTOMATED INTERPRETATION OFFluorescent Proteins and Fluorescence Resonance SUBCELLULAR LOCATION PATTERNS FROM THREE

Energy Transfer..................................................................794 DIMENSIONAL CONFOCAL MICROSCOPYQualitative Analysis ....................................................................795Preparation 795 Ting Zhao and Robert F Murphy

Nanobioscopy of Protein-Protein Interactions Introduction 818with Fluorescence Resonance Energy Transfer ..............795 Protein Subcellular Location .....................................................818Methods of Fluorescence Resonance Energy Transfer Overview of 2D Dataset Analysis ............................................818

Measurement...........................................................................795 High-Resolution 3D Datasets ..............................................820Sensitized Emission of Acceptor ..............................................795 3DHeLa.......................................................................................820Donor Fluorescence ................................................................... 796Acceptor Bleach..........................................................................797 I mage Acquisition Considerations When Using

Fluorescent Proteins as Fluorescence Resonance Automated Analysis...............................................................821Energy Transfer Pairs ........................................................ 798 Image Processing and Analysis 822Cyan Fluorescent Protein and Yellow Fluorescent

g

Segmentation of Multi-Cell Images andProtein - The Commonly Used Fluorescence Preprocessing..........................................................................822Resonance Energy Transfer Pair ..........................................798 3D Subcellular Location Features 822...............

Cyan Fluorescent Protein or Green Fluorescent Protein Automated Classification of Location Patterns................. 824Forms a Fluorescence Resonance Energy Transfer Classification of 3DHeLa Dataset ............................................824Pair with mRFPI .................................................................... 798 Downsampled Images with Different Gray Scales ................ 824

Fluorescence Resonance Energy Transfer-Based Clustering of Location Patterns: LocationSensors.....................................................................................798 Proteomics .................................................................................825

Fluorescence Resonance Energy Transfer and Other Exclusion of Outliers .................................................................825Complementary Methods............................................... 799 Determination of Optimal Clustering...................................... 825Fluorescence Resonance Energy Transfer and Statistical Comparison of Location Patterns ..................... 826

Fluorescence Lifetime Imaging Microscope .......................799 Image Database Systems.....................................................827Fluorescence Recovery After Photobleaching and Future Directions 827............................

Fluorescence Loss in Photobleaching ..................................801Fluorescence Resonance Energy Transfer and

Fluorescence Correlation Spectroscopy ...............................801 CHAPTER 48: DISPLAY AND PRESENTATIONFluorescence Resonance Energy Transfer and Total SOFTWARE

Internal Reflection Fluorescence..........................................801Quantum Dots and Fluorescence Resonance Energy Felix Margadant

Transfer...................................................................................801 Introduction..........................................................................829Cloning and Expression of Fluorescent Constructs Testing................................................................................... 830

for Fluorescence Resonance Energy Transfer ................ 801 "Static" Image Performance......................................................831Cloning of Fluorescent Chimeras .............................................801 Brightness....................................................................................832Functional Activity of Expressed Constructs ..........................802 Resolution: Changing the Display Size of YourExpression and Over-Expression ............................................. 802 Images..................................................................................... 832

Methods for Introducing Chromophores into Compression................................................................................835Living Cells........................................................................803 Motion Pictures....................................................................836Electroporation ........................................................................... 803 Coding Limitations 838

Up-Sampling or Frame Rate Matching ...................................838 Metadata Structure....................................................................867Motion Picture Artifacts ........................................................... 839 Digital Rights Management ..................................................... 867The MPEG Formats ..................................................................840 Future Prospects ..................................................................867MPEG Display Formats ........................................................... 840Very High Resolutions..............................................................841Movie Compression and Entropy............................................ 841 CHAPTER 51: CONFOCAL MICROSCOPY OFPerformance Benchmark.......................................................... 841 BIOFILMS - SPATIOTEMPORAL APPROACHESStoring Your Presentation for Remote Use .............................842Taking Your Presentation on the Road: Digital Rights R.J. Palmer, Jr., Janus A.J. Haagensen, Thomas R. Neu, and

Management and Overlaying...............................................844Claus Sternberg

Introduction.........................................................................870Sample Presentation ........................................................... 870

CHAPTER 49: WHEN LIGHT MICROSCOPE Flowcells and Other Perfusion Chambers ...............................870RESOLUTION IS NOT ENOUGH: CORRELATIONAL Water-Immersible Lenses .........................................................872LIGHT MICROSCOPY AND ELECTRON Upright Versus Inverted Microscopes ..................................... 872MICROSCOPY Setup of a Flow Chamber System Setup - A Practical

Paul Sims, Ralph Albrecht, James B. Pawky,Example................................................................................. 872

Victoria Centonze, Thomas Deerinck, and Jeff HardinMaking Bacteria Fluorescent ..............................................873

Fluorescent Proteins..................................................................873Introduction.........................................................................846 Stains ....................................................................................874

Early Correlative Microscopy..................................................846 Nucleic Acid Stains ...................................................................874Early 4D Microscopy ................................................................846 Live/Dead Stain.........................................................................875

Correlative Light Microscope/Electron Microscope Fluorescence In Situ Hybridization......................................... 875Today ................................................................................ 846 General Procedure for Embedding of Flowcell-GrownLight Microscope and Electron Microscope Have Biofilms for Fluorescence In Situ Hybridization ............... 876

Different Requirements ........................................................ 846 Antibodies ..................................................................................877Finding the Same Cell Structure in Two Different Preparation of Labeled Primary Antibodies ............................878

Types of Microscope: Light Microscope/Scanning Imaging Bacteria Without Fluorescence.................................879Electron Microscope............................................................. 850 I maging Extracellular Polymeric Substances

Finding the Same Cell Structure in Two Different in Biofilms..................................................................................879Types of Microscope: Light Microscope/Transmission Application of Two-Photon Laser-ScanningElectron Microscope............................................................. 852 Microscopy for Biofilm Analysis..................................... 882

Cryo-Immobilization Followed by Post-Embedding Limitations of Confocal Laser Scanning MicroscopyConfocal Laser Scanning Microscopy on Thin and Two-Photon Laser-Scanning Microscopy inSections..................................................................................856 Biofilm Analysis....................................................................884

Tiled Montage Transmission Electron Microscope Temporal Experiments........................................................ 885Images Aid Correlation.........................................................858 Time-Lapse Confocal Imaging.................................................885

Conclusion...........................................................................860 Summary and Future Directions........................................887

CHAPTER 50: DATABASES FOR TWO- AND CHAPTER 52: BIBLIOGRAPHY OF CONFOCALTHREE-DIMENSIONAL MICROSCOPICAL MICROSCOPYI MAGES IN BIOLOGY Robert H. WebbSteffen Lindek, Nicholas J. Salmon, and Ernst H.K. Stelzer A. Book and Review Articles .............................................889Introduction ......................................................................... 861 B. Historical Interest..........................................................889Data and Metadata Management in C. Theory (Mostly) .............................................................890

Microscopes.....................................................................861 D. Technical ........................................................................891Recent Developments............................................................... 861 E. General.......................................................................... 891

I mage Information Management ....................................... 862 F. Adaptive Optics.............................................................892The Aims of Modern Microscope System Design .............862 G. Differential.....................................................................892Instrument Database Model...............................................864 H. Display........................................................................... 892System Requirements ..........................................................864 I. Fiber-Optic Confocal Microscopes ............................. 893I mage Database Model.......................................................864 J. Index Mismatch.............................................................893Selected Projects ................................................................. 865 K. Multiplex ........................................................................894

Biolmage.................................................................................... 865 L. Nonlinear .......................................................................894Biomedical Image Library........................................................866 M. Polarization....................................................................894Scientific Image DataBase ........................................................866 N. Profilometry ...................................................................895Other Projects............................................................................ 866 O. Point Spread Function ..................................................895

Criteria and Requirements for Microscopy P. Pupil Engineering..........................................................896Databases ......................................................................... 866 Q. Thickness....................................................................... 896User Interface .............................................................................866 R. Turbidity.........................................................................896Query by Content ......................................................................866 S. Variants on the Main Theme........................................897

APPENDIX 1: PRACTICAL TIPS FOR APPENDIX 3: MORE THAN YOU EVER REALLY WANTEDTWO-PHOTON MICROSCOPY TO KNOW ABOUT CHARGE-COUPLED DEVICES

Mark B. Cannel!, Angus McMortand, James B. Pawkyand Christian .Sae/let Introduction 919................................Introduction.........................................................................900 Part I: How Charge-Coupled Devices Work .....................919

Laser Safety ..........................................................................900 Charge Coupling........................................................................919

Laser Alignment ...................................................................900 Readout Methods.......................................................................920

Testing Alignment and System Performance.....................900 What Could Go Wrong? ......................................................920

Laser Settings and Operation.............................................901 Quantum Efficiency...................................................................920

Monitoring Laser Performance ...........................................901 Edge Effects................................................................................921

Power Levels and Trouble -Shooting......................................903 Charge Loss................................................................................921Choice of Pulse Length.......................................................903 Leakage or "Dark Charge" ........................................................921Controlling Laser Power..................................................... 903 Blooming .................................................................................... 921Am I Seeing Two-Photon Excited Incomplete Charge Transfer......................................................923

Fluorescence or............................................................... 904 Charge Amplifiers.................................................................923Stray Light and Non-Descanned Detection...................... 904 What Is a Charge Amplifier? ....................................................923Laser Power Adjustment for Imaging at Depth.................904 FET Amplifier Performance ......................................................924Simultaneous Imaging of Multiple Labels......................... 904 Noise Sources in the Charge-Coupled Device.................924Minimize Exposure During Orientation and Fixed Pattern Noise................................................................... 924

Parameter Setting...................................................................905 Noise from the Charge Amplifier ............................................ 925Ultraviolet-Excited Fluorochromes.....................................905 Where Is Zero? ...........................................................................925

A New Idea: The Gain Register Amplifier!! ........................925Of Course, There Is One Snag! ................................................926

APPENDIX 2: LIGHT PATHS OF THE Part II: Evaluating a Charge-Coupled Device ...................927CURRENT COMMERCIAL CONFOCAL LIGHT A. Important Charge-Coupled Device Specs forMICROSCOPES USED IN BIOLOGY Live-Cell Stuff!................................................................927

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

Introduction.........................................................................906 Microscopy.......................................................................929BD -CARV II.................................................................................. 907 C. A Test You Can Do Yourself!!!..........................................930LaVision-BioTec TriM-Scope...............................................907 D. Intensified Charge-Coupled Devices .................................930Leica TCS SP2 AOBS-MPRS ................................................910Nikon C1si........................................................................... 911 Index.....................................................................................933Olympus Fluoview 1000-DSU............................................912Visitech VT Infinity-VT-eye................................................. 914Yokogawa CSU 22............................................................... 915Zeiss LSM-5-LIVE Fast Slit Scanner-LSM 510

META-FCS........................................................................916