9032 BioRad Confocal Inside/FIN...confocal microscopy and photon counting, and to determine actin...

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VISIBLE AND INVISIBLELASER RADIATION

AVOID EXPOSURE TO BEAMCLASS 3B LASER PRODUCT

LASER LIGHT - DO NOT STARE INTO

BEAM OR VIEWDIRECTLY WITH

OPTICALINSTRUMENTS

GLASS 3A LASER PRODUCTLASER OUTPUT FROM 400 TO 700nm

2.5W/cm2 MAXIMUM, C.W.

DANGERVISIBLE AND INVISIBLE LASER RADIATION

AVOID DIRECT EXPOSURE TO BEAM

CW LASER OUTPUT AT WAVELENGTHSIN THE RANGE OF 400 TO 700nm

MAXIMUM OUTPUT 25W m2

MODELOCKED LASER OUTPUT AT WAVE - LENGHTS IN THE RANGE OF 650 TO 1050nm

MAXIMUM AVERAGE POWER 1WPULSE WIDTH RANGE 80 - 200 fsec

REPETITION RATE 80 - 100 MHZ

CLASS lllb LASER PRODUCT

LASER RADIATION - DO NOT STARE INTO BEAM OR VIEW DIRECTLY WITH OPTICAL INSTRUMENTS

LASER OUTPUT AT WAVELENGHTSIN THE RANGE 400 TO 700nm2.5mW/cm2 MAXIMUM, C.W.CLASS llla LASER PRODUCT

Image Acknowledgments

Cover – left to rightJ. Song, Center for Research in Vascular Biology, Department of Anatomical Sciences, University of Queensland, Brisbane, AustraliaJ.B. Thomas, Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, USAR.R. Ribchester, T.H. Gillingwater and D. Thomson, Department of Neuroscience,University of Edinburgh, UK

Top - S. Jinno, Department of Anatomy and Neurobiology, Kyushu University, Japan Bottom- R. Anderson, Centre for Neuroscience and Department of Anatomy & Histology,Flinders University of South Australia, Australia

Page 2Top - Sample donated by A. Entwistle, Ludwig Cancer Research Institute, London, UKBottom - M. Baker, S.J. Rauth and E.R. Macagno, Department of Biological Sciences,Columbia University, New York, USA

Page 3Top-A. Fleury, M. Laurent and A. Adoutte, Universite Paris-Sud, FranceBottom – T. Deerinck, NCMIR, Department of Neurosciences,University of California San Diego, USA

Page 6J. Williams and S. Paddock, University of Wisconsin, USA

Page 7Left –A. Fleury, M. Laurent and A. Adoutte, Universite Paris-Sud, FranceRight – J. Beck, Department of Zoology, University of Washington, USA

Page 8Middle - R. Anderson, Centre for Neuroscience and Department of Anatomy & Histology,Flinders University of South Australia, AustraliaBottom- R.R. Ribchester, T.H. Gillingwater and D. Thomson, Department of Neuroscience,Universityof Edinburgh, UK

Page 16Calcium spikes - G. Giannone and C.D. Muller, Faculte de Pharmacie,Universite Louis Pasteur-Strasbourg, France

Page 18

T. Deerinck, J. Bouwer, S. Chow and M. Ellisman, NCMIR, Department of Neurosciences,

University of California San Diego, USA

Page 20Clockwise from top leftM. Apicella, Department of Microbiology, University of Iowa, USAR. Anderson, Centre for Neuroscience and Department of Anatomy & Histology,Flinders University of South Australia, AustraliaH. vanPraag, Laboratory of Genetics, The Salk Institute, La Jolla, USAJ. Song, Center for Research in Vascular Biology, Department of Anatomical Sciences,University of Queensland, Brisbane, Australia

Page 21FRET –A. Periasamy, W.M Keck Center for Celluar Imaging, University of Virginia, USAFREP – J. Wudel and N. Kikyo, Stem Cell Institute and Division of Hematology, Oncology andTransplantation, Department of Medicine, University of Minnesota, USA

Page 24Top left to bottom right2) R. Thazhath, University of Georgia, USA3) K.J. Daniels, D.C. Shutt and D.R. Soll, Department of Biological Sciences, University of Iowa, USA4) J. Song, Center for Research in Vascular Biology, Department of Anatomical Sciences, Universityof Queensland, Brisbane, Australia6) C. Cywes and M. Wessels, Brigham and Women’s Hospital and Children’s Hospitall, Boston MA7) M. Apicella, Department of Microbiology, University of Iowa, USA8) D. Ehrhardt , Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA9) R. Anderson, Centre for Neuroscience and Department of Anatomy & Histology, FlindersUniversity of South Australia, Australia10) K.J. Daniels, D.C. Shutt and D.R. Soll, Department of Biological Sciences, University of Iowa, USA12) H. vanPraag, Laboratory of Genetics, The Salk Institute, La Jolla, USA13) A. Fleury, M. Laurent and A. Adoutte, Universite Paris-Sud, France14) G. Lee and D. Gard, Department of Internal Medicine, University of Iowa, USA15) M. Köppen and J. Hardin, Cellular and Molecular Biology, University of Wisconsin, USA

Lissamine™ - trademark of Imperial Chemical IndustriesMicrosoft® - registered trademark of Microsoft CorporationCyDye™ - trademark of Amersham BiosciencesAll other ™ and ® - trademark/registered trademark of Molecular Probes Inc.

Radiance2100™ - Confocal Imaging Systems

Focused on

Being First

history

• Commercially available Confocal LSM.

• UV LSM.

• Dual channel system.

• Adjustable confocal aperture.

• LSM to offer photon counting detection.

• Multi line Krypton Argon laser in LSM.

• Time Course acquisition software in LSM

• Commercially available Multi-photon LSM

(MRC-1024 MP)

• Commercially available Blue Laser Diode

on an LSM.

• SELS (Signal enhancing lens) enabling

longer imaging of living material.

• Filter based spectral acquisition LSM

system (Radiance Rainbow).

Focused on Being FirstThe History of Bio-Rad Confocal SystemsBio-Rad has pioneered the field of Laser Scanning Microscopy (LSM). With a long history of product development and inno-vation, Bio-Rad building on over 18 years experience now offers the Radiance2100™, the latest offering based upon it’s 4thmajor generation of laser scaners.

Bio-Rad Laser Scanning Microscopy History

• MRC-500

• MRC-600

• DVC-250

• MRC-1000

• MRC-1024

• MicroRadiance

• RadiancePlus

• Radiance2000

• RTS2000

• Radiance2100

First in...

The Latest Generation

The Radiance2100 is the latest confocal laser scanningmicroscope (CLSM) from Bio-Rad, dedicated to the imaging ofbiological specimens. This represents the latest developments inthe 4th Generation of confocal systems, building on the successand innovation, which has lead to over 1700 installedinstruments and many thousands of publications.

From first pioneering the commercial confocal microscope in the early 80’s, Bio-Rad remains at the forefront of the field,using over 18 years of excellence to provide customers with astate of the art imaging tool. Made not only to give exceptionalresolution and sensitivity, the Radiance2100 is also designed togive users the flexibility to upgrade and incorporate additionalfunctionality.

Bio-Rad customers are not only able to benefit from this prestigious pedigree but the quality of instrument they receive,and also in the level and quality of service and support theycan expect having purchased a Bio-Rad Radiance.

Introduction to Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) allows researchersto image scattering fluorescent samples and to exclude out of focus light, creating stunning, high resolution images.

A laser beam provides the light for excitation and sophisticatedscanning optics allowing the sample to be imaged incombinations of x, y and z-axis (see diagram below).

Placing a confocal aperture in the emitted light path blocks the out of focus light so only the light that is in focus is collectedby the photomultiplier tubes (PMT’s).

This allows optical sectioning and the construction of 2D and 3D images, with the resulting images producing the ultimate influorescence LSM resolution.

Laser Scanning Microscopy

foreword 1

PMT Detector

Confocal Aperture

Beamsplitter

Laser

Sample

Objective Lens

9032 BioRad Confocal Inside/FIN 30/7/03 8:56 am Page 1

Scientific Collaborations

Bio-Rad has an active program of collaborations and technologylicensing with laboratories in several countries. In addition to licensingthe most innovative developments in optical design, there areongoing projects for the development of applications and instrumenttechnology. The Rank Prize (the Worlds most prestigious awardfor optoelectronics) highlights the success of such developmentsand was co-awarded to W.B. Amos, J.G. White, G.J. Brakenhoffand M. Minsky for the development of the confocal opticalmicroscope. In 2000 the Rank Prize was awarded to W. Denk andW. Webb, in recognition of their outstanding work on femtosecondpulsed lasers for multi-photon applications. W. Webb, J.G.White and W.B. Amos are consultants for Bio-Rad.

Bio-Rad Licensed Patents

Confocal Imaging System,

J. White 5,032,720

Achromatic Scanning System.

W.B. Amos US Patent 4,997,242

Multi-colour Laser Scanning

Confocal Imaging System.

Brelje Sorenson 5,127,730

Two-photon Laser Microscopy.

Denk, Strickler, Webb 5,034,613

Purchasing a confocal laser scanning system from Bio-Radrepresents the first step in, what we hope will be a long andrewarding association. We are committed to ensuring superiorcustomer care for all our products and to this end we providethe following:

• Remote access support

• A range of service contracts to suit differing needs and budgets

• Access to a team of experts in confocal microscopy

• On site service visits

• Telephone support

• Applications support for all users

“I can say that the service people that take care of mycontract have always been very helpful whenever wehave needed them. I have been very satisfied with thesystem since 1997 and it has worked almost daily”Marco A. M. Prado, Federal University of Minas Gerais, Brasil

Collaborations

Patents

collaborations and support2

ServiceQuality of service


Contents

Foreword 1

Collaborations and Support 2

Visibly Different 4

Optical Design 6

Sensitivity 8

Specificity 11

Spectral Detection 12

Versatility 16

Upgradeability 17

Compatibility 18

LaserSharp2000 19

Multi-Phase Time Course 21

Applications Resources 22

Configuration Chart 23

contents 3


Benefits To Users

• Retro-fittable to a range of upright and inverted microscopes.

• A wide range of laser options to cater the system to

your application.

• Upgradeable according to your needs

(including Multi-photon).

• Accurate separation and unmixing of overlapping emissionsignals with Radiance Rainbow.

• Variable confocal aperture for optimized spectral separation ineach channel.

• Maximal separation of signal with a choice of pseudo-bandpass filters, lambda strobing, and digital mixers (Real timespectral correction).

• Non-dispersive separation ensuring greater signal specificity.

• Live cell friendly through imaging with low laser power -increased signal with high sensitivity detectors, SensitivityEnhancement Lens (SELs) and Photon Counting.

Radiance2100™ - See the difference

visibly different4

The most sensitive confocal system80% of customers who have compared Bio-Rad

systems with others, claim that Bio-Rad confocals

produce the best quality images.


“The Bio-Rad Radiance 2100™ is extremely easy touse. Complete novices to confocal imaging can be upand running in a matter of minutes - there’s nothing Ihaven’t been able to do with the Radiance”Dr. Michael L. Koenig, WRAIR Walter Reed Army Institute of Research,Maryland, USA

1

43

21. BLD2. Scan Head3. Fast Z Drive4. ICU5. MCU

5

visibly different 5


Advanced manufacturing techniques for the best and reproducible performance

• Thermally matched components for repeatable performanceregardless of ambient conditions.

• Permanently aligned optics - no need for user adjustment.

• A unique optical design utilises infinity optics, a telescope andlarge variable, circular confocal apertures in each channel.

• Highly innovative point scanning design (US patent4,997,242, W.B. Amos) for reliable performance and thehighest image quality.

Proven Advanced Optics for LSM

“I used a Bio-Rad confocal to image Calcium by ratioconfocal microscopy and photon counting, and todetermine actin localization and organellar calciumlocalization in living cells. The technical features of Bio-Rad microscope enabled me to obtain highquality images which I published in JCS and JCB.”Lorelei Silverman-Gavrila, York University, Canada

Radiance2100™ Scanhead Overview

1. Polarisation preserving single-mode excitation fibre.

2. Achromatic Beamsplitter.

3. Fast galvos.

4. Concave mirror.

5. Telescope - to allow circular macro sized confocal apertures,

includes optional sensitivity enhancement lens

6. Dichroic chromatic reflectors.

7. Steering mirror.

8. Polarisers.

9. Confocal aperture and collector lens.

10. High sensitivity multi-mode emission fibre.

Only 2 channel Scanhead shown for simplicity

optical design6


Ideal scanning geometry for un-compromisedimage quality

In the Radiance2100 scanhead both x and y-scan motions areproduced by fast galvanometer-type mirrors using a uniquedesign. Ideal scanning geometry is achieved by using twoconcave mirrors to perfectly image one galvanometer on the other.This produces rotation about a point on the second mirror and theback aperture of the objective is filled throughout the completescan cycle. This is crucial with respect to maximising detectionsensitivity and image uniformity.

Scanning system with two close-coupled galvo mirrors. The oscillation of thefirst mirror (blue dotted) produces beam movement on the second. Of thethree successive beams shown, only the central ones enters the back pupil ofthe objective lens correctly.

A non ideal system. The diagram shows the inferior close-coupled galvo design. Inthis, the back focal plane is only partly filled at the ends of the scan, unless the beamis made so large that it overfills the aperture. The return beam (not shown) has anundesirable side -to-side translation during the scan, which has to be compensatedfor by complex optics.

7optical design

first galvo mirror

second galvo mirror


Prismatically Enhanced-Photo-Multipliers

• Prismatically enhanced PMTs (Unique to Bio-Rad) causeinternal reflection of the photons at the photomultiplier window and increases quantum efficiency, doubling the sensitivity of conventional PMTs.

• Enables use of low laser power - Ideally suited to live cellimaging applications.

• PMTs are situated in the ICU, thus minimising thermaleffects in order to keep the dark current and image noiselow without noisy cooling mechanisms - for betterquality images.

• High sensitivity PMT’s are selected for optimal performancein each channel.

Outstanding Detection Sensitivity

QE Comparison of PMT (S20) with & without Enhancer Prism

Wavelength (nm)

Qu

an

tum

Eff

icie

ncy

(%)

Standard PMT

Enhanced PMT

sensitivity8


Motorised Sensitivity Enhancement Lens (SELS)

• Effectively increases the size of the detector aperture significantly, capturing the light scattered by a sample thatwould otherwise be lost at the confocal aperture.

• Signal intensity is typically increased by a factor of five ormore allowing much lower laser powers.

• Reduced photo-toxicity giving significant advantages duringextended live imaging procedures such as expression ordevelopmental studies.

• Optional for Multi-photon and visible laser systems. Usefulfor when it is acceptable to trade confocality for sensitivity.

Photon Counting

• Digitally accumulates the real number of photons counted; one photon gives one pulse.

• Does not accumulate noise.

• A particularly powerful detection mode for extremely faintsamples e.g. low transfection GFP, or autofluorescence.

• Allows less laser power and reduces photo-toxicity.

• Provides the best signal to noise ratio detection available.

• Extremely sensitive – counts up to 30 photons within a single pixel.

• Excellent linearity of response, temperature stability and insensitivity to PMT voltage fluctuations ideal for quantitative studies.

Stomata cells with FITC, DAPI and Chlorophyll. When imaged in analoguemode, noise accumulation is noticed (top). When imaged using photoncounting mode only true signal is accumulated (bottom).

sensitivity

Neuronal sample imaged with optimal iris (top) and then using half of thelaser power with iris open to five times optimal (middle) and with optimaliris and the SELS lens (bottom).

9


Optimised Transmission Detection, DIC and Reflection Imaging

• Optimised input fibres to retain the polarisation properties of the laser beam.

• Dedicated polarisers and reflection filters per channel .

• DIC sensitivity is as high as video-enhanced DIC.

High Sensitivity PMT Transmission Detector

• Two eight position filter wheels for blocking and emission filters.

• Enables DIC imaging with much lower laser power.

Fully AdjustableConfocal Apertures - Control for real lifeexperiments

• Radiance2100 featuresfully adjustable circularapertures to provide fullcontrol over opticalsectioning at allwavelengths.

• Independent variableaperture per channel.

• Flexible control forimaging differentintensity fluorophores in each channel insimultaneous scanning.

• Essential for highsensitivity live sampleimaging where highlaser power would bedamaging, confocalitycan be traded for signalstrength.

• Unlike systems that utilise microscopic apertures there is no need for regular re-alignment of the optical path.

Technical note 9 “Optimum optical design characteristics for confocal and

multi-photon imaging systems” by W.B. Amos, MRC Cambridge ,UK.

A human cheek cell imaged with a photo-diodedetector (left) and the PMT detector (right). Thephoto-diode image required 30% laser power(488 nm), whilst the PMT image required only 3%laser power (488 nm). This enables the collectionof a high contrast DIC image at the same time as afluorescence image, using typical (2-5%) laserexcitation power.

sensitivity

circular aperture

square aperture

10

Radiance2100™ CircularAperture Overview

A comparison of the variable, circularaperture used in the Radiance2100 with thesquare aperture, piezo driven apertures usedin other systems. The Radiance2100aperture opens to 12mm and is drawn inproportion to the square aperture.


Dichroics and Emissions Filters - High ResolutionSpectral Separation Performance Every Time

• The use of an achromatic primary beamsplitter ensures highefficiency transmission of excitation wavelengths andcollected fluorescence across the visible spectrum withoutthe signal fall off associated with excitation dichroics. Inaddition the use of an achromatic beamsplitter allowsadditional lasers to be added without changing thebeamsplitter.

• Dichroic beamsplitters and emission filters provide convenientpermutations for fluorophore selection.

• Emission filters have 100% reproducible spectral resolution,which unlike existing spectrometer based selection methods,is independent of the beam diameter or the selected spectral region.

• Filters ensure steep spectral cut-offs, of selected wavelength range.

• The filters supplied will cover commonly used fluorophoresbut for specialist work users may substitute their own emissionfilters or use a Radiance2100 Rainbow system.

YOYO®-1, Cy3™, Cy5™: TheYOYO®-1 is bleeding through

Bleed through correctedusing RTSC

High Specificity

High Quality 500 - 560nm Filter

Wavelength (nm)

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

450 500 550 600

Transm

issi

on

specificity

Real Time Spectral Correction (RTSC)

• Unique to Bio-Rad.

• Allows digital subtraction of fluorescence bleed throughfrom the image in real time, resulting in pure images.

• Live measurements allow correction before the sample deteriorates.

• Uncorrected images can be captured for reference or analysis by other means.

• Easily controlled in LaserSharp2000 software.

Lambda Strobing

Lambda strobing sequentially excites the sample line by line with different laser lines and is a highly effective approach tocorrecting bleed-through.

11

Simultaneous imaging

Lambda strobing


The new Rainbow model of the Radiance2100 uses a broadseries of filter combinations to allow users to configure theoptimal bands to perform spectral separation. This uniqueapproach maintains all the functionality and sensitivity of theRadiance2100 system.

The Rainbow system also includes the new SpectraSharpspectral reassignment software, allowing users to spectrallyreassign overlapping fluorophores into pure channels.

The Separation Issue

Previously, discrete emission filters have been used to collectspectral bands of the emission signal. The advantage of filters isthat they enable very high levels of signal to be captured. Inaddition, unlike dispersive optics spectral resolution remainsconstant for all confocal aperture sizes. To separateoverlapping fluorophores, the following techniques areavailable on non-Rainbow Radiance systems.

• Careful selection of fluorophores.

• Frame by frame sequential excitation.

• Line by line sequential excitation (Lambda strobing).

• Real Time Spectral Correction using the Digital mixers (RTSC).

Although these techniques allow clearly separated acquisition

of most fluorophores, may not be sufficient for:

• Significantly overlapping fluorescent proteins - CFP, GFP, YFP & DsRed.

• Auto-fluorescent signals overlappingfluorophore emission.

• Unavoidable co-excitation.

Flexible Band-pass Filters

The Radiance2100 Rainbow utilises advances in filtertechnology, which allow filters with sharp spectral propertiesto be used across the whole imaging spectrum.

• Combining long pass and short pass filters it is possible tocreate over 10000 band permutations.

• Optimally configured for the collection of fluorescence withsimilar and overlapping emission.

• Maintains separate gain and confocal aperture controls foreach channel, allowing the balancing of different intensityfluorophores without damaging the sample.

Full Sensitivity Spectral DetectionFrom near UV to IR using Proven Technology

Emission Wheel

Blocking Wheel

PMT

The Radiance2100 Rainbow Detection System

spectral detection12

“The images I obtained were highly appreciated byreviewers and I received invitations to submit picturesfor the journal covers.”

Rosalind Silverman-Gavrila, York University, Canada


1 2

Images spectrally reassigned using SpectraSharp

1. GFP and strong auto fluorescence.

2. Spectrally reassigned image with GFP as green and auto fluorescence as red.

spectral detection 13

FITC / Texas Red ScatterPlot Before (top) and after(right) reassignment.

(Actual images shown on right)

Original merge (above) and new merge of reassigned images (below).

Pure FITCReference line

Pure Texas Red®

Reference line

SpectraSharp -Spectral Reassignment Software

When collection of pure channels is simply not possible usingtraditional techniques or the Rainbow detection flexibilitythe SpectraSharp software performs Spectral Reassignmentto create separated images. SpectraSharp does not require the collection of a spectral series to perform spectralreassignment. This enables users to collect and process images easily and quickly.


14 spectral detection

Ultimate Performance

• Highly flexible filter combinations.

• Characterise fluorescence response.

• Non-dispersive to ensure spectral resolution is maintainedregardless of confocal aperture size.

• Spectral Reassignment without the need to collect spectral series.

• Compatible with Multi-Photon, Photon Counting and the SELS.

• Maintain ultimate signal to noise ratios.

The example above shows a triple labelled sample with three fluorescentproteins:CFP, GFP and YFP. 1. Natural colours, 2. raw images collected by Radiance Rainbow, 3. images unmixed with SpectraSharp.

Focal Check™ - Double GreenFluorescent Micro spheres (F-36905from Molecular Probes). Outer layerhas 500nm excitation / 512nmemission. Inner core has 512nmexcitation / 525nm emission.

A two channel z-series of the Microspheres was collected simultaneouslywith 488nm excitation; image shownis a spectrally reassigned sideprojection with the outer layer onlyrendered every three sections.

1 2

3


15

Spectral Series

• Collect series of images in 10nm steps.

• Plot graphs corresponding to the emission caused by a laser line.

• Enables optimal filter configuration to be determined forfluorophores with unknown properties.

The Achromatic Beamsplitter

All Radiance systems use an Achromatic primary Beamsplitter. Byusing advances in filter design the polarised laser light is diverted tothe sample whilst returning emission is collected with much higherlevels of efficiency compared to traditional beamspliters.

• Laser light diverted utilising polarisation properties insteadof wavelength – removing the need for dips intransmission curve.

• Over 81% efficiency at all wavelengths between 400 and 700nm

• Factory mounted to maintain these efficiency levels without theneed for user or engineer realignment

• No moving parts removing chance of pixel shift when scanning sequentially, allowing accurate co-localisation andspectral reassignment.

• Not susceptible to thermal effects

• Ideal for all scan modes including simultaneous andlambda strobing

• Compatible with scattered light detection for Multi-photonimaging

• Future proof for new laser lines

• Future proof for new fluorophores

• Use lower laser powers for less photo-bleaching

• Increased signal to noise ratios allowing better images andfaster scan rates

Achromatic Beamsplitter represented by blue curve, versus traditional beamsplitterwith red curve. Achromatic comparative losses shown in dark red and gains shown ingreen. In this example the Achromatic beamsplitter gives 173.3% efficiency of CFPemission collection and 158.5% efficiency of GFP emission collection compared to thetraditional beamsplitter.

Image showingregions of interestused to create thespectral graph.

spectral detection


16

Scan Modes to Suit a Variety of Applications

• x, y, z, t combinations including: xy, xyt, xt, xz, xyz, xyztscans to user defined parameters.

• Simultaneous, frame by frame sequential or line by linesequential (lambda strobing) acquisition.

• Direct scanning, kalman integration filtering to reduce noise or accumulate filtering for weak signals.

• Optical Panning, Zooming and 360 degree scan rotation Scans.

• Automated vertical sectioning (50nm step resolution) allows Z-series to be collected to produce 3D rendering projections and animations.

• Multi-phase time course (optional) allows acquisition, playback and live plotting of data with different imaging conditions over time. e.g. drug studies, calcuim imaging,bleaching etc.

Acousto Optical Tuneable Filter (AOTF)Modulation for Precise Scanning Control

• Controls the mix of excitation wavelengths and the relative power of each wavelength from the laser(s) enabling high spectral separation, even in simultaneousmulti-channel acquisition.

• Operates interactively from the laser control panel in LaserSharp2000.

• Fly-back blanking cuts off the excitation illumination duringthe return part of the galvo scanning cycle to significantlyreduce photo-bleaching.

• Provides ultra fast laser switching for Region of Interest (ROI) scanning.

Fast Imaging Suited for Physiology/Live CellStudies

• Minimised time loss between frames for maximum signal to noise ratios.

• Fast scanning in uni and bi-directional scanning modes combined with minimised dead time allow frame rates over 100fps.

• Data transferred with IEEE 1394 (FireWire™) eliminating needfor data compression and allowing real time display of data.

• Synchronisation signals are output by the Radiance2100every frame, line and pixel.

• Scanning can be triggered with only a 4ms delay.

• Image hard marking allows external events to be recordedwith the image with 1 pixel accuracy.

CAIRN Integrator

• Combines up to 7 channels of analogue physiologicalrecordings with the optical information.

• Complete confidence in the synchrony between the datasets.

• Allows indefinite scans, recorded directly to hard drive.

Versatility

versatility

Calcium spikes inastrocytoma cells


17

The Radiance2100 has been specifically designed to suit theneeds of the user. Bio-Rad understand that the needs andpriorities of customers change with time. To this end, numerousupgrade options are made available to users of the Radiance2100. These are designed to allow the inclusion of additionaloptions to provide additional functionality, meaning that withRadiance, users always have the right tool for the job.

First Blue Laser Diode 405nm (BLD™)

• Ideal for imaging DAPI, CFP, Hoechst, Alexa Fluor™430,Alexa Fluor™ 405, KAEDA, PA-GFP and a wide range ofnear-UV fluorophores.

• Does not subject your sample to the damaging effects of UV.

• Optimised co-excitation ratio’s for CFP/YFP FRET.

HeCd and Kr Secondary Lasers

Additional laser lines for better fluorophore excitation:

The Ergonomic Manual Control Unit (MCU)

The MCU is an ergonomic unit with large distinctive controls for easy touch sensitive identification. This allows optimisation of all of the essential image acquisition parameters without having to avert your eyes from the screen and is particularlyuseful when acquiring many images.

• One touch start/stop.

• Easy fine control of imaging parameters.

• Use default or user defined configurations.

• Real time response toadjustments.

Upgradeability

Krypton 568nm HeCd 442nm

Texas Red® CFP

Alexa Fluor™ 568 Cameleon

DsRed Fura Red™

Cy3™ Lucifer Yellow

MitoTracker™ Red LysoTracker® Green

LysoSensor™ Red Chlorophyll

Lissamine™ Rhodamine

upgradibility


Motorised XY Stages

Bio-Rad offers a variety of xy stages adapting to most microscopes and offering:

• Sub-micron resolution and repeatability.

• Joystick contriol for ease of movement.

Software control provides the following functionality:

• ‘Bookmarking’ to record and return positions of interest.

• Repeat visits to sites during automated acquisition, i.e. 3D or 4Dacquisition at multiples locations.

• Tiling and stitching of multiple 2D or 3D images to create large true highresolution images.

Microscope Compatibility

Radiance2100 enables researchers to use a wide range of microscopes. Its compatibility with existing units can reducethe need for additional investment.

Models in italics are compatible with multi-photon, models in boldhave motorised control of key functions like light path, focus control andobjective lens position from the LaserSharp2000 acquisition software.

Upright Microscopes

Nikon: E600, E600FN, E800, E1000, Optiphot 2,

Olympus: BX50, BX50WI, BX51, BX51WI, BX60, BH2,

Zeiss: Axioplan, Axioplan2, Axioplan2 Imaging, Axioskop1, Axioskop2,AxioskopFS, Axiotech, Axiophot 1 & 2,

Inverted Microscopes

Nikon: TE300, TE2000-S, TE2000-U, TE2000-E, DV, Diaphot 300, Diaphot TMD

Olympus: IX70, IX71, IX81

Zeiss: Aviovert100/135, Axiovert 35M, Axiovert 200

If you have more than one scope that you would like touse the Radiance2100 on it is possible to switchbetween microscopes in minutes.

Compatibility

compatability18


19LaserSharp2000

The Suite of Laser Scanning MicroscopySoftware

With over 18 years of experience manufacturing laser scanningmicroscopes, there are two features that we strive to incorporateinto all of our software - usability and power. LaserSharp2000is the core package for image acquisition and the more commonlyused image processing features.

Operating Software for the Radiance and MRC-1024 Laser Scanning Microscopes

Our 4th generation acquisition package LaserSharp2000 is optimised for ease of use. The prominent graphical control panellets users easily access the most common acquisition features.User accounts store individual preferences for files saving andacquisition, allowing you to start scanning with one click when

you return to the system. The optics diagram allows users tosee a visual representation of their system and to change theoptics even whilst the system is scanning.

LaserSharp2000™


Core - Image Processing Functions

Arithmetic operations, histograms, line profiles, seed-fill, colocalization and merge. Projections can be produced with a wide range of visualisation algorithms including ‘Two-Pass’ projections to add opacity to 3D views.

Refractive Index Compensation

LaserSharp2000 provides automaticcompensation for objective lenseswith different numerical aperturesand media with different refractiveindices. Z-axis focal shift is virtuallyeliminated and hence the 3D imagesyou acquire are free from distortion.

Simple and Powerful Scripting

In LaserSharp2000, Microsoft® VBScript is used to provide thefacility for users to write macros or scripts. Scripting facility include:

• Access to software and firmware functions.

• Instrument control functions.

• Data manipulation/processing.

• Programmable status bar, user interface, progress bars andoutput windows.

Users can add functionality, automate and customise systemoperation. An online library of scripts is available to downloadand swap pre-written scripts.

Example of just a few of the functions easily performed with scripting:

• Ratio metric imaging.

• Logarithmic laser power increment through very thick Z-series to compensate for signal drop off.

• Image processing including monitoring and responding tointensity changes through series.

• Synchronisation of external peripherals to acquisition.

• Automate FRET aquisitions.

The Bio-Rad PIC File Format

• Standard loss less image format – standardised since the MRC-500.

• Supported by most third party software.

• Stores all collection parameters within PIC file for future reference or to return system to same imaging conditions.

• Retains spatial, spectral and temporal calibration without effort.

Additional software options for 2D/3D/4D studies,co-localisation and deconvolution are also available. Pleaseask your sales representative for more details on these.

LaserSharp200020


multi phase time course 21

• Third generation time course package developed by Bio-Rad to allow user to perform multiphase time course experiments.

• Configure collection parameters for multiple phases and run through the phases in order.

• Experiments can be paused, or skipped to the next phase atany time.

• Regions of interest can be drawn on the image and the intensities plotted in real time.

• By combining multiphase time course with the patterned Illumination, or zoom to region of interest functionality of the Radiance2100, FRAP experiments can be conducted by bleaching a region for one phase and measuring fluorescence recovery in the next phase.

Fluorescence Resonance Energy Transfer (FRET)

Justin Wudel and Nobuaki Kikyo - Stem Cell Institute and Division of Hematology, Oncologyand Transplantation, Department of Medicine, University of Minnesota, USA - App Note 37.

Confocal FRET Microscopy

Localization of CFP- and YFP-C/EBPa protein expressed in mouse 3T3 cells. The doubly expressed cells (CFP-YFP-C/EBPa ) were excited by donor excitationwavelength and the donor (D) and acceptor (UFRET) images of proteins localized in the nucleus of a single living cell were acquired. FRET data analysis algorithmwere used to obtain the processed PFRET image. (Elangovan et al., 2002a).Confocal: donor Ex: 457nm Em: 485/30nm

acceptor Ex:514nm Em: 545/40nm FRAP of GFP-B23 in a HeLa cell: The GFP signal is shown in the four steps ofFRAP. The square area was photobleached and the fluorescence recovery wasmonitored. Bar indicates 5µmm.

Multi-Phase Time CourseIdeal for FRAP and FRET

“Bio-Rad Radiance2100 is a truly wonderful confocalmicroscopy system for the time course experiment, such as FRAP and FLIP. It is easy to use for inexperienced people but the image quality is superb. Its versatile featuresupported by the knowledgeable technical staff makes the system an ideal imaging tool for us. ”

Nobuaki Kikyo MD PhD, Assistant Professor, University of Minnesota

Fluorescence Recovery After Photobleaching (FRAP) Example – Detection of nucleolar proteinB23 shuttling

Dr. Nobuaki Kikyo and his colleagues at the University ofMinnesota are studying dynamic disassembly and reassembly ofthe nucleolus in the context of somatic nuclear cloning in HeLacells. They are using the FRAP technique to better understandkinetics of association and dissociation of individual proteins inthe nucleolus. They describe the movement of GFP-taggednucleolar protein B23 using the FRAP technique with confocalimaging.

Quantitative Analysis of FRAP

Fluorescence intensity in the prebleach image was defined as1.0; all other values are relative to this intensity.


application resources22

Support for your applications @www.cellscience.bio-rad.com

Bio-Rad Cell Science Division offers not only quality productsto help with your research, but also applications support andscientific resources to help you use these products. We areexpanding our applications support on the web, which currentlyincludes frequently asked questions, application and technicalnotes, image gallery, interactive fluorescence database, usefulfluorophore and academic links, and a reference (bibliography)source, to name a few.

The image gallery contains a plethora ofspectacular images coveringmany application areas, with detailed image descriptionsand source references.This can help you place alaboratory doing similarresearch.

Application and Technical Notesdetail specific applications that you can refer to, as well as technical details of system features. You can have your interesting research featured in one of these notes.

Fluorophore Resource

• Fluorophore data - A comprehensive but non-exhaustive list of fluorophores (alphabetically divided), providing excitation and emission maxima for over 600 fluorophores.

• Interactive fluorophore database – Plan your experiments with a “An Excellent Resource”

Launched in May 1999, this website has proven to be a greatsuccess with scientists all around the world with feedback that isgreatly appreciated. Since the beginning of last year alone, wehave had over 24,000 users of this facility, of which 8000 userswere new visitors!

This easy-to-use web page features an online help to guide first-time users through this powerful tool. You can plot the excitationand emission profiles of up to 4 fluorochromes at once, and thenoverlay laser lines and filter data. The database can be searched

by name or by user defined bandwidth of excitation or emissionwavelengths. This tool can help you plan your experiments fromanywhere before you visit the confocal saves you from losingprecious microscope time!

Fluorophore Wall Chart

The Fluorophore Wall Chart is designed to give the user “at aglance” fluorophore data. Both the excitation and emissionmaxima(nm) are shown on this attractive wall chart, along withsome spectacular images taken by our friends around the worldwith Bio-Rad systems.

This tool is designed to aid the user in the selection of the correctfluorophore for use with their samples and microscope, and willbrighten the wall of anylaboratory!

If you would like to orderyour copy of the newFluorophore wall chart, orsubmit YOUR spectacularimages please visit ourweb site.

Applications Resources

“Very informative”Howard Seliger, McCollum Pratt Institiute and Department of Biology,

The Johns Hopkins University

“This program is an excellent resource - I use it often!” Kathy Turner, PhD candidate, North Carolina State University

“Very effective and easy to use”Gerald A. Campbell, M.D., Ph.D. University of Texas Medical Branch

Colored graphs are

plotted and ready

for printing.


23

Spectral Reassignment

Time Course

Image Acquisition s/w

2D Image Analysis s/w

3D Rendering s/w

Computer

Signal Enhancement Lens

High Sensitivity PMT



Spectral Detection

Motorised Stage (microscope specific)

Fast Z Drive

2 Channel Direct Detectormotorised/manual

4 Channel Direct Detectormotorised/manual

2/4 Channel Direct DetectorFLIM ready option

Filter blocks

Multi Photon Laser

Collimation Lens

Beam Attenuation

Beam Conditioning Unit (BCU)

Argon Laser457, 477, 488 and 514nm

Blue Diode Laser405nm

HeCd442nm

Green HeNe Laser543nm

Krypton Laser568nm

Red Diode Laser637nm

Manual Control Unit

Transmission Detector

High SensitivityTransmission Detector

N D Pockel Cell

Fixed Motorised

Manual Upright/InvertedMicroscope

Motorised Upright/InvertedMicroscope

Scanhead

Visible System

sM

ulti

Pho

ton

Sys

tem

s

InstrumentControl Unit

(ICU)

Krypton Argon Laser488/568nm


ISO 9001 reg is tered b io- rad.com

Cel l Sc ience D iv is ion

9MRC60BR20

As part of our policy of continual improvement, Bio-Rad reserve the right to change technical specifications without notice

www.cellscience.bio-rad.comBio-RadLaboratories

VISIBLE AND INVISIBLELASER RADIATION

AVOID EXPOSURE TO BEAMCLASS 3B LASER PRODUCT

LASER LIGHT - DO NOT STARE INTO

BEAM OR VIEWDIRECTLY WITH

OPTICALINSTRUMENTS

GLASS 3A LASER PRODUCTLASER OUTPUT FROM 400 TO 700nm

2.5W/cm2 MAXIMUM, C.W.

DANGERVISIBLE AND INVISIBLE LASER RADIATION

AVOID DIRECT EXPOSURE TO BEAM

CW LASER OUTPUT AT WAVELENGTHSIN THE RANGE OF 400 TO 700nm

MAXIMUM OUTPUT 25W m2

MODELOCKED LASER OUTPUT AT WAVE - LENGHTS IN THE RANGE OF 650 TO 1050nm

MAXIMUM AVERAGE POWER 1WPULSE WIDTH RANGE 80 - 200 fsec

REPETITION RATE 80 - 100 MHZ

CLASS lllb LASER PRODUCT

LASER RADIATION - DO NOT STARE INTO BEAM OR VIEW DIRECTLY WITH OPTICAL INSTRUMENTS

LASER OUTPUT AT WAVELENGHTSIN THE RANGE 400 TO 700nm2.5mW/cm2 MAXIMUM, C.W.CLASS llla LASER PRODUCT

Image Acknowledgments

Cover – left to rightJ. Song, Center for Research in Vascular Biology, Department of Anatomical Sciences, University of Queensland, Brisbane, AustraliaJ.B. Thomas, Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, USAR.R. Ribchester, T.H. Gillingwater and D. Thomson, Department of Neuroscience,University of Edinburgh, UK

Page 1Top - S. Jinno, Department of Anatomy and Neurobiology, Kyushu University, Japan Bottom- R. Anderson, Centre for Neuroscience and Department of Anatomy & Histology,Flinders University of South Australia, Australia

Page 2Top - Sample donated by A. Entwistle, Ludwig Cancer Research Institute, London, UKBottom - M. Baker, S.J. Rauth and E.R. Macagno, Department of Biological Sciences,Columbia University, New York, USA

Page 3Top-A. Fleury, M. Laurent and A. Adoutte, Universite Paris-Sud, FranceBottom – T. Deerinck, NCMIR, Department of Neurosciences,University of California San Diego, USA

Page 6J. Williams and S. Paddock, University of Wisconsin, USA

Page 7Left –A. Fleury, M. Laurent and A. Adoutte, Universite Paris-Sud, FranceRight – J. Beck, Department of Zoology, University of Washington, USA

Page 8Middle - R. Anderson, Centre for Neuroscience and Department of Anatomy & Histology,Flinders University of South Australia, AustraliaBottom- R.R. Ribchester, T.H. Gillingwater and D. Thomson, Department of Neuroscience,Universityof Edinburgh, UK

Page 16Calcium spikes - G. Giannone and C.D. Muller, Faculte de Pharmacie,Universite Louis Pasteur-Strasbourg, France

Page 18

T. Deerinck, J. Bouwer, S. Chow and M. Ellisman, NCMIR, Department of Neurosciences,

University of California San Diego, USA

Page 20Clockwise from top leftM. Apicella, Department of Microbiology, University of Iowa, USAR. Anderson, Centre for Neuroscience and Department of Anatomy & Histology,Flinders University of South Australia, AustraliaH. vanPraag, Laboratory of Genetics, The Salk Institute, La Jolla, USAJ. Song, Center for Research in Vascular Biology, Department of Anatomical Sciences,University of Queensland, Brisbane, Australia

Page 21FRET –A. Periasamy, W.M Keck Center for Celluar Imaging, University of Virginia, USAFREP – J. Wudel and N. Kikyo, Stem Cell Institute and Division of Hematology, Oncology andTransplantation, Department of Medicine, University of Minnesota, USA

Page 24Top left to bottom right2) R. Thazhath, University of Georgia, USA3) K.J. Daniels, D.C. Shutt and D.R. Soll, Department of Biological Sciences, University of Iowa, USA4) J. Song, Center for Research in Vascular Biology, Department of Anatomical Sciences, Universityof Queensland, Brisbane, Australia6) C. Cywes and M. Wessels, Brigham and Women’s Hospital and Children’s Hospitall, Boston MA7) M. Apicella, Department of Microbiology, University of Iowa, USA8) D. Ehrhardt , Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA9) R. Anderson, Centre for Neuroscience and Department of Anatomy & Histology, FlindersUniversity of South Australia, Australia10) K.J. Daniels, D.C. Shutt and D.R. Soll, Department of Biological Sciences, University of Iowa, USA12) H. vanPraag, Laboratory of Genetics, The Salk Institute, La Jolla, USA13) A. Fleury, M. Laurent and A. Adoutte, Universite Paris-Sud, France14) G. Lee and D. Gard, Department of Internal Medicine, University of Iowa, USA15) M. Köppen and J. Hardin, Cellular and Molecular Biology, University of Wisconsin, USA

Lissamine™ - trademark of Imperial Chemical IndustriesMicrosoft® - registered trademark of Microsoft CorporationCyDye™ - trademark of Amersham BiosciencesAll other ™ and ® - trademark/registered trademark of Molecular Probes Inc.

Radiance2100™ - Confocal Imaging Systems

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