Holdings: Confocal microscopy for biologists

Confocal microscopy for biologists:

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Bibliographic Details
Main Author:	Hibbs, Alan R. (Author)
Format:	Book
Language:	English
Published:	New York [u.a.] Kluwer Acad.,Plenum Publ. 2004
Subjects:	Confocale microscopie Microscopie confocale Confocal microscopy Microscopy, Confocal Konfokale Mikroskopie Biologie
Online Access:	Inhaltsverzeichnis
Physical Description:	XIII, 467 S. zahlr. Ill., graph. Darst.
ISBN:	0306484684

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adam_text	Confocal Microscopy for Biologists Alan R Hibbs BIOCON Melbourne, Australia Kluwer Academic / Plenum Publishers New York, Boston, Dordrecht, London, Moscow Contents 1 WHAT IS CONFOCAL MICROSCOPY? OPENING NEW AREAS FOR LIGHT MICROSCOPY MULTI-SKILLED ENDEAVOUR LASER SCANNING CONFOCAL MICROSCOPY Epi-fluorescence Illumination How is the Image Formed? How is Out of Focus Light Removed? Optical Slicing NIPKOW DISK CONFOCAL MICROSCOPY MULTI-PHOTON MICROSCOPY IMAGING CAPABILITIES Fluorescence Imaging Time-lapse Imaging 3Dimensional Information JUST FANCY IMAGES - OR A NOVEL APPROACH TO BIOLOGY? Live Cell and Tissue Imaging Biochemistry of Single Cells Applications of Confocal Microscopy WHY USE A CONFOCAL OR MULTI-PHOTON MICROSCOPE? Advantages Limitations 2 UNDERSTANDING MICROSCOPY ESSENTIAL OPTICS Wave Nature of Light Interacting Light Waves Refraction and the Bending of Light Rays Counting Photons BASIC MICROSCOPY Image Formation using a Simple Lens Compound Microscope Numerical Aperture (NA) Importance of the Condenser Immersion Media Coverslip Thickness IMAGING MODES Bright-field Imaging Transmission Imaging Phase Contrast Imaging DIC (Nomarski) Imaging — Backscatter Imaging Fluorescence Imaging OPTICAL ABERRATIONS Chromatic Aberration Spherical Aberration Coma Astigmatism Curvature of Field Optical Aberrations Caused by the Specimen Resolution in a Light Microscope COMPONENTS OF A MICROSCOPE Microscope Layout Optical Components Objectives Eyepieces (Oculars) Beam Splitter Optical Filter Block (or Optical Filter Wheel) Condenser Condenser Iris 60 Field Aperture 60 Focus Controls 61 Microscope stage 61 Microscope Attachment Ports 61 Confocal Microscope Scan Head Attachment Port 61 Camera Attachment Port 61 Fibreoptic Transmission Pickup 62 Light Sources 62 Mercury Arc Lamp 63 Xenon Arc Lamp 63 Tungsten Lamp 63 Halogen Lamp 63 3 CONFOCAL MICROSCOPY HARDWARE 65 INSTRUMENT DESIGN 65 LASER SCANNING CONFOCAL MICROSCOPES 66 Instrument Layout 66 Irradiation of the Sample 70 Scan Head and Associated Optics 71 Scanning Mirrors 71 Confocal Pinhole (Iris) 72 Optical Filter Separation of Fluorescent Light 73 Acoustic Optical Beam Splitter (AOBS) 78 Hardware Settings for Dual Labelling 79 Spectral Separation of Fluorescent Light 81 Lasers used in Confocal Microscopy 86 Widely used Lasers 86 Laser Line Selection 88 Attenuation of Laser Light 88 Light Detectors 89 Photomultiplier (PMT) Tubes 90 Photodiode Light Detectors 90 Instrument Control Panel 90 NIPKOW DISK CONFOCAL MICROSCOPES 91 Nipkow Disk 91 Micro-Lens Array 93 Light Sources for Nipkow Disk Confocal Microscopy 93 CCD Camera Detectors 93 MULTI-PHOTON MICROSCOPES 94 Microscope Design 95 Pulsed Infrared Lasers 95 Direct (External) Detectors 97 CONFOCAL MICROSCOPE MANUFACTURERS 97 WHICH CONFOCAL MICROSCOPE SHOULD I USE? 99 4 IMAGE COLLECTION 101 IMAGE COLLECTION MODES 102 Fluorescence Image Collection 102 Backscatter (Reflectance) Image Collection 107 Transmission Image Collection 108 Real Colour Transmission Image Collection 110 Photon Counting 110 LASER INTENSITY AT THE SAMPLE 110 Laser Power Level 111 Laser Intensity Level 112 INSTRUMENT SENSITIVITY CONTROL 112 Gain Level or Photomultiplier Tube (PMT) Voltage 113 ix x Contents Black Level or Offset Control Digitisation PINHOLE ADJUSTMENT SCAN SPEED Why have Different Scan Rates? Is there an Optimal Scan Speed? Decreased Scan Speed - for Improving your Image Slow Scanning for Live Cells Fast Scanning for Moving the Sample Fast Scanning to Catch Movement Bi-directional Scanning COLLECTION FILTERS Continuous Single Frame Image Collection Single Frame Capture Mode Line Averaging Screen Averaging •Caiman Averaging Collection to Peak Exponential Collection IMAGE COLLECTION BOX SIZE CONFOCAL MICROSCOPE ZOOM Scanning Zoom Panning While Zoomed Zooming for Optimal Resolution Over Sampling and Under Sampling Digital Zooming after Image Collection REGION OF INTEREST (ROI) SCANNING IMAGE COLOUR Using Colour to Optimise Image Collection OPTIMAL IMAGE COLLECTION Understanding Noise in an Image Sources of noise 3D IMAGE COLLECTION Extended Focus Image Rocking Motion Image Red/Green Stereo Pair Offset Stereo Pair TIME RESOLVED IMAGE COLLECTION Is THERE A STANDARD SAMPLE LOOKING AFTER YOUR IMAGES Filing your Images Choosing a Unique Naming System Managing your images Data Backup 5 DIGITAL IMAGES IN MICROSCOPY IMAGE PIXELS ~ — COMPUTER SCREEN RESOLUTION Screen Dots (dpi) Display Resolution COLOUR IN DIGITAL IMAGES Human Perception of Colour Computer Colour Generation Colouring your Image Colour Look Up Tables (LUTS) Colour 24-bit (RGB) Images Colour in Merged Images Printing Colour IMAGE MANIPULATION Contrast Stretching Image Enhancing Filters QUANTITATIVE ANALYSIS OF IMAGES PIXELS Image Histograms FILE FORMATS 6 IMAGING SOFTWARE 163 IMAGE COLLECTION SOFTWARE 164 IMAGE MANIPULATION AND ANALYSIS SOFTWARE 164 Imaging Software from Confocal Microscope Manufacturers 165 Basic Image Manipulation Software 165 Software for Presentation and Preparation of Figures 166 Sophisticated 2D and 3D Image Manipulation Software 166 Software for Archiving Images 167 Confocal Assistant (free software) 168 Photoshop 173 CorelDRAW 175 PowerPoint 176 7 PRESENTATION AND PUBLICATION 177 HONESTY IN IMAGING RESEARCH 177 Some Points to Remember when Presenting Imaging Results 179 PRODUCING PUBLICATION QUALITY IMAGES 180 Collecting Top Quality Original Data 181 Collect Images in Grey-Scale 181 Enhancing your Image for Publication 181 MAKING CONVENTIONAL SLIDES 181 Slides from a Computer Screen 181 Colour Slide Makers 182 USING A COMPUTER PROJECTOR 182 PowerPoint Presentations 182 SUBMISSION OF FILES FOR PUBLICATION 183 PRINTING FOR PUBLICATION 183 Inkjet Printers 184 Black and White Laser Printer 184 Colour Laser Printers 184 Dye Sublimation Printers (Colour and Grey-Scale Printing) 184 Printing on Photographic Paper 185 8 WHAT IS FLUORESCENCE? 187 WAVELENGTHS OF LIGHT 187 EXCITATION OF A FLUOROPHORE 188 Single Photon Excitation 188 Multi-photon Excitation 189 FLUORESCENCE EMISSION SPECTRA 190 Environmental Influence on Fluorescence Spectra 191 BRIGHTNESS OF A FLUOROPHORE 191 WHICH EXCITATION WAVELENGTH IS BEST? 192 MOLECULAR STRUCTURE OF COMMON FLUOROPHORES 194 Fluorescein, the Green Fluorescent Dye 194 Tetramethylrhodamine (TMR), Red Fluorescent Dye 195 Alexa Fluor Dyes 195 PHOTOBLEACHING 196 Controlling Photobleaching in Fixed Samples 196 Controlling Photobleaching in Live Cells 197 AUTOFLUORESCENCE 198 Autofluorescence in Living Cells and Tissues 198 Fluorescence Created by Fixation 199 Using Autofluorescence to Visualise Cell Structure 199 CHOOSING THE CORRECT DYE 200 9 FLUORESCENT PROBES 201 WHAT ARE FLUOROPHORES FOR? 201 GETTING INFORMATION ON FLUOROCHROMES 202 Internet 202 Molecular Probes Catalogue and Internet Site 202 Bio-Rad Fluorescence Spectra Internet Site 202 Confocal Microscopy Listserver Archive Site 203 Information from Flow Cytometry can be Very Useful 204 WHAT FLUORESCENT PROBES ARE AVAILABLE? 204 REACTIVE FLUOROPHORES 204 —,147 Contents xi Traditional Fluorescein (FITC), TMR and Texas Red Dyes 206 Alexa Fluor Dyes 208 Cyanine Dyes (Cy2, Cy3 and Cy5) 210 NUCLEIC ACID PROBES 212 Acridine Orange (AO) 212 Propidium Iodide (PI) 213 Ethidium Bromide (EthBr) 214 UV DNA Dyes (DAPI, Hoechst 33258 etc) 214 SYTO Dyes (cell permeant) 214 SYTOX Dyes (do not enter live cells) 214 FLUORESCENT ION INDICATORS 216 How do Ion Indicators Work? 216 Esterase Derivatives 217 Ca2t Indicators 218 pH Indicators 221 Membrane Potential Probes 222 Other Ion Indicators 222 ORGANELLE PROBES 223 Mitochondria Specific Probes 223 Lysosomal Probes 226 Golgi Probes 226 Endoplasmic Reticulum Probes 226 MEMBRANE PROBES 227 CELL TRACERS 228 CELL INTEGRITY 230 SOME UNUSUAL FLUORESCENT PROBES 230 PhiPhiLux (Apoptosis marker) 230 Latex Beads 231 Quantum Dots 231 GREEN FLUORESCENT PROTEIN (GFP) 232 Structure of GFP 232 Enhanced Derivatives of GFP 233 Coloured Variants of GFP 233 GFP from the Sea Pansy (Renilla reniformis) 233 Fluorescent Proteins from Coral (Discosoma sp) 234 Light Sources for GFP Excitation 234 Photoactivation and Photoconversion GFP 234 GFP Applications 235 GFP Limitations 236 FLASH - HEXA-PEPTIDE FLUOROPHORE TAG 238 10 CONFOCAL MICROSCOPY TECHNIQUES239 MULTIPLE LABELLING 240 Choice of Fluorophore 240 Labelling the Cells 240 Multi-Channel Collection 241 Minimising Cross-Talk between Channels 242 Determining the Degree of Co-localisation 244 COMBINING FLUORESCENCE AND TRANSMISSION IMAGES 249 Merging Images - Photoshop Layers 249 Merging Images - Photoshop Channels 251 FRAP, FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING 252 FRET, FLUORESCENCE RESONANCE ENERGY TRANSFER 254 FISH, FLUORESCENCE IN SITU HYBRIDISATION 256 LINE SCANNING 256 QUANTITATIVE CONFOCAL MICROSCOPY 256 INFORMATION ON CONFOCAL MICROSCOPY TECHNIQUES 257 Bio-Rad Application and Technical Notes 257 11 FLUORESCENCE IMMUNOLABELLING 259 WHAT IS IMMUNOLABELLING? 259 Using a Confocal Microscope for Immunolabelling 261 LABELLING METHODS 263 Indirect Immunolabelling 263 Direct Immunolabelling 264 Dual Immunolabelling 266 Biotin/Streptavidin Immunolabelling 266 PREPARATION OF SAMPLES FOR IMMUNOLABELLING 267 Fixation with Organic Solvents 268 Fixation with Aldehyde Fixatives 269 Microwave Fixation 270 Commercially Available Fixative Preparations 270 Permeabilisation 270 Embedding Tissue Samples in ParafTin Wax 271 Cryo-sectioning of Tissue Samples 271 PRACTICAL HINTS FOR IMMUNOLABELLING 272 Application of Your Antibody 272 Using a Humidity Chamber 273 Washing to Remove Excess Antibody 273 Application of Secondary Antibody 273 Blocking of Non-specific Background Labelling 274 Preparation of Slides for Microscopy 274 AUTOFLUORESCENCE IN IMMUNOLABELLED SAMPLES 275 Immunofluorescence Antibody Labelling (IFA) Method 276 IMMUNOLABELLING OF LIVE CELLS 277 Cell Surface Labelling 277 Intemalisation of Cell Surface Receptors 277 Streptolysin-0 (SLO) Permeabilised Cells 277 12 IMAGING LIVE CELLS 279 MAINTAINING HEALTHY CELLS 280 Keeping Cells Alive 280 Dangers of Stressing the Cells 281 Apoptosis - Programmed Cell Death 282 Recognizing Signs of Cell Stress 282 SIMPLE IMAGING CHAMBERS 283 Traditional Concave Microscope Slide 283 Simple Gasket Chamber 284 Microscope Slide Chamber 284 Sterile Culture Chambers 286 Simple Flow Chamber 287 ATTACHMENT OF CELLS 288 Attaching Suspension Cells to the Coverslip 288 Growing Attached Cells for Microscopy 289 TEMPERATURE CONTROL 290 Why is Temperature Control Important? 291 Is Temperature Control Needed while Imaging? 293 Temperature Control on Transfer to Microscope 294 TEMPERATURE CONTROL CHAMBERS FOR MICROSCOPY 295 Simple Temperature Control Chambers 296 Heated Objectives 296 Heated Microscope Enclosures 297 Home Made Reflective Plastic Microscope Enclosure 298 Problems with Microscope Enclosures 298 Heated Microscope Stages 301 Petri Dish Warming Platforms 301 Heated Microscope Slide Holders 303 Perfusion Chambers 304 Bioptechs Chamber 308 MAINTAINING THE CORRECT PHYSIOLOGICAL STATE 311 Oxygen Level 311 pH Control 311 Osmolality of the Media 312 Long-Term Growth 312 LIVE TISSUE SAMPLES 313 Tissue Chambers 313 Maintenance of Live Tissue 314 Dye Penetration 314 Imaging Depth 315 DYE LOADING INTO LIVE CELLS 315 Lipid Soluble Dyes 315 Methyl Ester Derivatives of the Dye 316 xii Contents Microinjection Transfection Semi-permeabilisation using Streptolysin-O (SLO) Introducing Reagents while Imaging THE CORRECT LENS FOR LIVE CELL IMAGING Low Magnification Dry Lenses Oil Immersion High NA Lenses Water Immersion Lenses Dipping Lenses MINIMISING LASER DAMAGE What is Photo Damage? Antifade Reagents Laser Power AUTOFLUORESCENCE IN LIVE CELLS As a Probe for Cellular Structure and Function Controlling Unwanted Autofluorescence IMAGE COLLECTION TIPS FOR LIVE CELLS High Speed Collection Specialised High Speed Confocal Microscopes Multi-photon Imaging of Live Cells Slow Scan Collection Single-Line Collection 13 THE INTERNET CONFOCAL MICROSCOPY LISTSERVER WEB SITES RELEVANT TO CONFOCAL MICROSCOPY International Mailing Lists Useful Research and Core Facility Sites Courses on Fluorescence Imaging amp; Confocal Microscopy Confocal Microscope Manufacturers Supply Companies Imaging Software Microscope Imaging Chambers 14 TECHNICAL SUPPLIES CONFOCAL MICROSCOPES IMAGING SOFTWARE FLUORESCENT PROBES AND ANTIBODIES GREEN FLUORESCENT PROTEIN AND DERIVATIVES MICROSCOPY EQUIPMENT AND REAGENTS OPTICAL FILTERS SIMPLE MICROSCOPY CHAMBERS MICROSCOPY PERFUSION CHAMBERS MICROSCOPE HEATERS TEMPERATURE CONTROLLED MICROSCOPE STAGES TEMPERATURE CONTROLLEDIMAGING CHAMBERS FLOW CHAMBERS 15 FURTHER READING OPTICS MICROSCOPY CONFOCAL MICROSCOPY CONFOCAL MICROSCOPY APPLICATIONS FLUORESCENCE GREEN FLUORESCENT PROTEIN (GFP) HISTOLOGY AND SAMPLE PROCESSING CELL BIOLOGY TECHNIQUES IMAGE PROCESSING APPENDIX 1: CONFOCAL MICROSCOPES 355 Laser Spot Scanning Confocal Microscopes 355 Nipkow Spinning Disk Confocal Microscopes 355 Nipkow Disk / Micro-lens Array Confocal Microscopes 355 A BIO-RAD CELL SCIENCE DIVISION 356 BIO-RAD INSTRUMENTS 356 RADIANCE SERIES OF CONFOCAL MICROSCOPES 358 Components of the Radiance Confocal Microscopes 359 Radiance Scan Head 360 Radiance Instrument Control Unit (ICU) 362 Changing Optical Filters and Dichroic mirrors 363 Radiance 2100 Rainbow 363 SELS (Signal Enhancing Lens) 364 Photon Counting 364 Computer and Software 364 MRC SERIES OF CONFOCAL MICROSCOPES 365 Components of the MRC-1024 Confocal Microscope 366 MRC-1024 Scan Head 367 Scan Head Alignment 368 Optical Filter Blocks 369 Emission Filter Wheels 372 Photomultiplier Tubes (PMT) 372 Controller Box 372 Computer Requirements 372 Hardware set up for specific imaging methods 372 Dual and Triple labelling - T1 and T2A Filter Blocks 373 Live Cell Work - B1 and OPEN BLOCK Filter Blocks 374 Back Scatter Imaging - B1 and T1 Filter Blocks 37S TROUBLE SHOOTING (MRC AND RADIANCE) 376 BIO-RAD LASERSHARP SOFTWARE 377 Image Collection Panel 378 Main Control Panel 379 Optics Control Panel 380 Mixer Control Panel 381 CARL ZEISS MICROSCOPY 382 ZEISS CONFOCAL MICROSCOPES 382 ZEISS LSM 510 META CONFOCAL MICROSCOPE 384 Components of the Zeiss 510 META 385 Zeiss LSM 510 META Scan Head 386 Lasers 386 Single Track and Multi Track Image Collection 388 Conventional Detection Channels 388 META Channel 388 Transmission Imaging 390 ZEISS CONFOCAL MICROSCOPE CONTROL SOFTWARE 391 Main Control Panel 391 Setting up the Microscope and Imaging Channels 392 Setting the Scan Control Parameters 393 Adjusting Imaging Channel Settings 394 Image Collection Panel 395 Using the META Channel 396 META Channel as a multi-Band Pass Optical Filter 396 META Channel - Lambda Mode (Spectral Imaging) 397 Extracting Image Channels from a Spectral Stack 397 Emission Fingerprinting 400 On-Line Emission Fingerprinting 400 LEICA MICROSYSTEMS 402 LEICA CONFOCAL MICROSCOPES 402 Components of the Leica Spectral Imaging Instrument 404 Leica TCS SP2 Confocal Microscope 405 Leica TCS SP2 Scan Head 405 Acoustic Optical Beam Splitter (AOBS) 406 Contents xiii Spectral Separation 406 UV Excitation 407 Multi-Photon Excitation 407 Transmission (DIC) Imaging 407 LEICA CONFOCAL MICROSCOPE CONTROL SOFTWARE 408 Main Control Panel - Monitor 1 408 Changing the Desktop Knob Functions 410 Beam Path Setting Control Panel 411 Control Buttons for Image Collection 412 Image Collection Panel - Monitor 2 414 NIKON INSTRUMENTS 415 NIKON CONFOCAL MICROSCOPES 415 Components of the Nikon CI Confocal Microscope 417 Nikon CI Scan Head 418 Optics/Detector Unit 418 Laser Launch Unit 418 NIKON Cl USER INTERFACE 419 OLYMPUS CORPORATION 420 OLYMPUS CONFOCAL MICROSCOPES 420 Components of the Olympus Fluoview 1000 422 FLUOVIEW 1000 CONFOCAL MICROSCOPE 423 SIM (Simultaneous) Scanner 424 FLUOVIEW 500 CONFOCAL MICROSCOPE 424 Fluoview 500 Scan Head 424 Components of the Olympus Fluoview 300 and 500 425 FLUOVIEW 300 CONFOCAL MICROSCOPE 427 Fluoview 300 Scan Head 427 Fluoview Software 428 Image Collection Panel 428 Main Control Panel 429 Instrument Settings Tabs 430 ATTO BIOSCIENCE 431 ATTO BIOSCIENCE CONFOCAL MICROSCOPES 431 CARV CONFOCAL MICROSCOPES 432 Components of the CARV Confocal Microscope 433 CARV Scan Head 434 YOKOGAWA ELECTRIC CORPORATION 435 CSU10 SCAN HEAD 435 CSU21 SCAN HEAD 437 PERKINELMER LIFE SCIENCES 438 PERKINELMER CONFOCAL MICROSCOPES 438 Ultra VIEW LCI layout 439 Components of the PerkinElmer Ultra VIEW 440 VISITECH INTERNATIONAL 441 VISITECH CONFOCAL MICROSCOPES 441 GLOSSARY 444 INDEX 461
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publisher	Kluwer Acad.,Plenum Publ.
record_format	marc
spelling	Hibbs, Alan R. Verfasser aut Confocal microscopy for biologists Alan R. Hibbs New York [u.a.] Kluwer Acad.,Plenum Publ. 2004 XIII, 467 S. zahlr. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Confocale microscopie gtt Microscopie confocale Confocal microscopy Microscopy, Confocal Konfokale Mikroskopie (DE-588)4336446-9 gnd rswk-swf Biologie (DE-588)4006851-1 gnd rswk-swf Konfokale Mikroskopie (DE-588)4336446-9 s Biologie (DE-588)4006851-1 s DE-604 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012862494&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Hibbs, Alan R. Confocal microscopy for biologists Confocale microscopie gtt Microscopie confocale Confocal microscopy Microscopy, Confocal Konfokale Mikroskopie (DE-588)4336446-9 gnd Biologie (DE-588)4006851-1 gnd
subject_GND	(DE-588)4336446-9 (DE-588)4006851-1
title	Confocal microscopy for biologists
title_auth	Confocal microscopy for biologists
title_exact_search	Confocal microscopy for biologists
title_full	Confocal microscopy for biologists Alan R. Hibbs
title_fullStr	Confocal microscopy for biologists Alan R. Hibbs
title_full_unstemmed	Confocal microscopy for biologists Alan R. Hibbs
title_short	Confocal microscopy for biologists
title_sort	confocal microscopy for biologists
topic	Confocale microscopie gtt Microscopie confocale Confocal microscopy Microscopy, Confocal Konfokale Mikroskopie (DE-588)4336446-9 gnd Biologie (DE-588)4006851-1 gnd
topic_facet	Confocale microscopie Microscopie confocale Confocal microscopy Microscopy, Confocal Konfokale Mikroskopie Biologie
url	http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012862494&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT hibbsalanr confocalmicroscopyforbiologists

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