Holdings: Handbook of biological confocal microscopy

Handbook of biological confocal microscopy:

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Bibliographic Details
Format:	Book
Language:	English
Published:	New York [u.a.] Plenum Press 1995
Edition:	2. ed.
Subjects:	Confocale microscopie Confocal microscopy Microscopy, Confocal Biologie Mikroskopie Biowissenschaften Konfokale Mikroskopie Konferenzschrift > 1989 > San Antonio Tex.
Online Access:	Inhaltsverzeichnis
Item Description:	Literaturangaben
Physical Description:	XXIII, 632 S. Ill., graph. Darst.
ISBN:	0306448262

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245	1	0	\|a Handbook of biological confocal microscopy \|c ed. by James B. Pawley
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adam_text	Contents CHAPTER 1: FOUNDATIONS OF CONFOCAL SCANNED IMAGING IN LIGHT MICROSCOPY Shinvu Inoue Light microscopy .................................. Lateral resolution ................................ Axial resolution ................................ Depth of field ................................ Confocal imaging............................... Impact of video ................................ Nipkow disk ................................. Electron-beam-scanning TV..................... Impact of modern video ........................ Lasers and microscopy .......................... Holography .................................. Laser illumination............................. Laser-illuminated confocal microscopes........... Confocal laser-scanning microscope .............. Is laser-scanning confocal microscopy a cure-all? . . Speed of image or data acquisition ............... Depth of field in phase-dependent imaging......... Other optical and mechanical factors affecting confocal microscopy Lens aberration ............................... Unintentional beam deviation ................... Contrast transfer and resolution in confocal vs. nonconfocal microscopy...................... Summary...................................... 3 3 4 5 5 6 6 7 7 7 8 9 10 10 II 13 14 14 CHAPTER 2: FUNDAMENTAL LIMITS IN CONFOCAL MICROSCOPY James Pawley Introduction ...................................... 19 What limits? .................................... 19 Counting statistics: The importance of«.............. 19 Source brightness ................................ 20 Specimen response: Dye saturation .................. 20 A typical problem................................ 21 Practical photon efficiency 22 Losses in the optical system........................ 22 Detection and measurement losses................... 25 Where have all the photons gone . 28 Resolution: How much is enough? 30 Can resolution be too high? ........................ 31 Limitations imposed by spatial and temporal quantization 32 Practical considerations relating resolution to distortion . 34 Conclusion ....................................... 36 CHAPTER 3: QUANTITATIVE FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY (CLSM) David R Sandison, Rebecca M Williams. K Sam Hells. James Stnckler. and Walt W Webb Introduction.................................. Confocal rejection of out-of-focus background ... Background rejection for alternative geometries ... Spatial filtering for ideal confocal resolution compromises signal collection ................. Optical transfer efficiency (MRC-600 1993) Effect of optical aberrations on fluorescence CLSM . Chromatic aberrations in fluorescence CLSM measurements................................ Photodynamic effects............................ Theory ...................................... Fluorescence emission rates ..................... Experiment................................... Fluorescence photobleaching recovery with CLSM . Conclusion .................................... Summary...................................... 39 40 43 43 44 45 46 47 47 47 49 50 51 51 CHAPTER 4: THE PIXILATED IMAGE Robert H Webb and C Kathleen Dorev Introduction ..................... 55 1 ixclation in the human eye .................... 55 Pixelation ...................................... 55 Resel: The optical resolution element ................ 57 Pixel: The picture element......................... 57 In between ..................................... 57 Intensity resolution............................... 57 Gray level...................................... 57 Matching image spatial characteristics............... 58 The Nyquist Theorem ............................ 58 Hazards of undersampling aliasing .................. 59 The resel/pixei ratio......,....................... 60 Monitors....................................... 60 Hyper-resolution: Ovcrsampling.................... 60 The mechanics of pixelation ....................... 61 Matching image intensity characteristics ............. 62 Gray scale...................................... 62 Display ........................................ 63 Caveats ........................................ 64 Strategy for magnification and resolution ............ 66 Summary......................................... 66 Take-home lessons ................................ 66 xvi Contents CHAPTER 5: LASER SOURCES FOR CONFOCAL MICROSCOPY Enrico Gratton and Martin J. vandeVen Introduction...................................... 69 Laser power requirements.......................... 69 The basic laser.................................... 70 Principle of operation ............................ 70 Pumping power requirements ...................... 71 Laser modes: Longitudinal (axial) and transverse ...... 71 Polarization .................................... 71 Coherent properties of laser light ................... 72 Heat removal ................................... 73 Other installation requirements..................... 73 Attenuation of laser beams ........................ 73 Stabilization of intensity, wavelength, and beam position in lasers ............................... 73 Sources of noise in lasers.......................... 73 Beam-pointing improvement via active laser cavity stabilization.................................. 75 Beam delivery and positioning ..................... 75 Optical tweezers................................. 75 Spatial beam characteristics........................ 76 Types of lasers.................................... 76 Continuous-wave lasers........................... 76 Pulsed lasers.................................... 81 Wavelength expansion through nonlinear techniques .. 83 Second and higher harmonic generation ............. 83 Sum or difference mixing ......................... 84 Optical parametric oscillators (OPO) and amplifiers (OPA) ...................................... 84 Trends in time-resolved spectroscopy applied to microscopy .................................... 84 Gain-modulated image intensifiers .................. 85 Two-photon excitation............................ 85 Maintenance ..................................... 85 Maintenance of active laser media .................. 85 Maintenance of pumping media .................... 86 Maintenance of the optical resonator ................ 86 Maintenance of other system components ............ 86 Safety precautions ................................ 87 Conclusion....................................... 87 Appendix A. CW gas lasers......................... 91 Appendix B. Pulsed laser systems ................... 95 Appendix C. Sample listing of manufacturers ......... 97 CHAPTER 6: NON-LASER LIGHT SOURCES Victor Chen Introduction: Not using laser light for confocal microscopy and how to use laser light when it s all you have ...................................... 99 Wavelength .................................... 99 Coherence...................................... 101 Types of confocal microscopes that can use non-laser sources...................................... 101 Characteristics of non-laser light sources............. 101 Source radiance ................................. 102 Source stability ..................................102 Source coherence ................................103 Source distribution............................... 103 Collecting the light and relaying it to the specimen ___103 Illumination of the specimen .......................103 Tandem scanning, basic description..................103 Single-sided disk scanning, basic description ..........105 Optical design of the illumination system .............105 Scrambling and filtering the light....................106 Measuring what comes through the illumination system . 107 Exposure time and source brightness.................107 What if the specimen is moving or changing?..........107 How to use laser light if it s all you have..............108 Conclusion .......................................108 Useful addresses...................................108 CHAPTER 7: OBJECTIVE LENSES FOR CONFOCAL MICROSCOPY H. Ernst Keller Introduction ......................................Ill Aberrations of refractive systems ....................112 Defocusing .....................................112 Monochromatic aberrations ........................113 Chromatic aberrations.............................118 Finite vs. infinity optics.............................122 Working distance..................................123 Optical materials ..................................124 Antireflection coatings .............................124 Transmission of microscope objectives ...............125 Conclusion .......................................125 CHAPTER 8: THE SPECIMEN ILLUMINATION PATH AND ITS EFFECT ON IMAGE QUALITY Carol J. Cogswell and Kieran G. Larkin Introduction ......................................127 Optical components and layout of the illumination path .. 127 Purpose of the illumination path.....................127 Properties of the illuminating spot ...................127 Illuminating spot size and shape.....................128 Maximum irradiance of the illuminating spot ..........128 Defects that may occur in the illumination path .......129 How to measure or observe defects in the illumination path.........................................129 Locating and correcting defects in the illumination path .131 Summary .........................................137 CHAPTER 9: THE INTERMEDIATE OPTICAL SYSTEM OF LASER-SCANNING CONFOCAL MICROSCOPES Ernst H. K. Stelzer Introduction ......................................139 Design principles of confocal systems ................139 Overview.......................................139 Microscope objectives ............................139 Contents xvii Position of the pivot point .........................140 Position of the detector pinhole .....................141 Filling the back focal plane ........................ 141 Practical requirements .............................142 Illumination..................................... 142 Detection.......................................142 Distortion ...................................... 143 Evaluation of illumination/detection systems..........143 Influence of optical elements on the properties of light .. 143 Errors caused by optical elements ................... 143 Evaluation of optical arrangements .................. 144 Evaluation of scanner arrangements .................146 Z-scanners......................................148 Disk scanners ...................................148 Object scanners..................................149 Attachment to microscopes ........................149 Merit functions ..................................149 Fluorescence experiments ..........................150 Special optical elements............................150 Multimode optical glass fibers......................150 Single-mode, polarization-preserving glass fibers ......150 Polarizing elements...............................151 Mechanical scanners..............................151 Mechanical patterns ..............................152 Acousto-optical scanners ..........................152 Vibration isolation ...............................152 Commercial instruments ...........................152 Conclusions.......................................153 CHAPTER 10: INTERMEDIATE OPTICS IN NIPKOW DISK MICROSCOPES G. S. Kino The tandem scanning reflected light microscope (TSM) 155 Other real-time scanning optical microscopes......... 155 Images of the eye.................................. 158 z-resolution....................................... 59 Pinhole size....................................... 161 Pinhole spacing ................................... 63 Illumination efficiency and reflection from the disk 164 Internal reflections ................................ 164 Conclusions....................................... 164 Summary......................................... 165 CHAPTER 11: THE ROLE OF THE PINHOLE IN CONFOCAL IMAGING SYSTEM T. Wilson Introduction ...................................... 167 The theory of optical sectioning property............. 167 Finite, circular detector, and coherent light............ 168 Lateral resolution as a function of effective detector size ........................................... 171 The role of aberrations ............................ 171 Images with a finite-sized detector .................. 172 Extended-focus and auto-focus imaging .............. 173 Height imaging.................................. 173 Alternative detector geometries..................... 174 Sensitivity to flare................................. 176 Fluorescence imaging.............................. 177 Alternative kinds of pinhole ........................ 180 Compact confocal microscopy using laser feedback ... 181 Conclusions ...................................... 181 Summary......................................... 182 CHAPTER 12: PHOTON DETECTORS FOR CONFOCAL MICROSCOPY Jonathan Art Introduction...................................... 183 The quantal nature of light ......................... 183 Interaction of photons with materials................ 184 Thermal effects ................................. 184 Direct effects ................................... 184 Photoconductivity ............................... 184 Photovoltaic .................................... 184 Photoemissive .................................. 186 Comparison of detectors .......................... 187 Noise internal to detectors ......................... 187 Noise in internal detectors......................... 187 Noise in photoemissive devices .................... 188 Statistics of photon flux and detectors ............... 189 Representing the pixel value ....................... 190 Conversion techniques............................. 191 Assessment of devices ............................. 192 Point detection assessment and optimization .......... 192 Field detection assessment and optimization .......... 194 Detectors present and future ....................... 195 CHAPTER 13: THE COLLECTION, PROCESSING, AND DISPLAY OF DIGITAL THREE-DIMENSIONAL IMAGES OF BIOLOGICAL SPECIMENS Hans Chen, Jason R. Swedlow, Marcus Grote, John W. Sedat, and David A. Agard Introduction...................................... 197 Data collection ................................... 197 Image processing ................................. 198 CCD camera calibration .......................... 199 Data errors ..................................... 199 Photobleaching.................................. 200 Deblurring by constrained iterative deconvolution ..... 200 Multiple-wavelength image alignment............... 201 Image enhancement............................... 201 Linear filters.................................... 201 Median filters................................... 202 Local contrast enhancement ....................... 202 Edge enhancement by gradient ..................... 202 Extracting information from volumetric data sets ..... 203 3D graphics .................................... 203 Volume rendering ............................... 203 Display methods for biological data sets ............. 205 Stereo display................................... 205 Movie display................................... 206 xviii Contents Image overlay .................................... 207 Data measurement by model building ............... 207 A software system for all three-dimensional microscopy applications......................... 207 Implementation methods and features ............... 208 Summary ........................................ 209 CHAPTER 14: VISUALIZATION SYSTEMS FOR MULTIDIMENSIONAL CLSM IMAGES N. S. White Introduction...................................... 211 Definitions ..................................... 211 What is the microscopist trying to achieve?........... 211 Criteria for choosing a visualization system........... 211 Why do we want to visualize multidimensional CLSM data?.......................................... 213 Data and dimensional reduction .................... 213 Objective or subjective visualization? ............... 214 Identifying unknown structures..................... 214 Highlighting previously elucidated structures ......... 214 Visualization for multidimensional measurements ..... 214 What CLSM images can the visualization system handle? ....................................... 214 Image data: How are image values represented in the program? .................................... 214 Dimensionality: What dimensions can the images and views have?.................................. 215 Standard file formats for calibration and interpretation . . 223 Processing image data ............................ 223 Processor performance—How fast will my computer process images? .............................. 223 How will the system generate the reconstructed views? 224 Assessing the four basic steps in the generation of reconstructed views ........................... 224 Loading the image subregion ...................... 224 Choosing a view: The five-dimensional image display space ....................................... 225 Mapping the image space into the display space ....... 228 How do 3D visualizations retain the Z-information? .... 233 Mapping the data values into the display ............. 237 How can intensities be used to retain Z-information? ... 240 Hidden object removal............................ 243 Adding realism to the view ...................... 244 How can I make measurements using the reconstructed views? ........................... 250 Conclusions ...................................... 250 Appendix: Details of visualization systems and other suppliers ...................................... 253 Digital video disk systems for reconstruction animations, etc.......................................... 254 Stereo viewers, stereo presentations, etc.............. 254 CHAPTER 15: MAPPING AND MEASURING SURFACES USING REFLECTION CONFOCAL MICROSCOPY Alan Boyde and Sheila J. Jones Introduction ......................................255 Mechanical focus mapping.........................255 Recovery of data from recorded stereo-pairs...........255 Chromatic aberration mapping and imaging ...........255 Nonconfocal methods for imaging and mapping 3D surfaces........................................256 Scanning electron microscopy and stereophotogrammetry 256 Interference methods..............................257 Scanned-tip microscopies..........................257 Confocal through-focus mapping ....................257 Choice of instrumentation..........................257 Biological microsurveying examples .................258 Measurement of the function of bone-resorbing cells .... 258 Measurement of cell volumes in vitro ................260 Characterization of tooth wear......................261 Practical focus mapping and measurement............261 Data acquisition..................................261 Segmenting the MAP .............................261 Missing data zones and the choice of NA .............261 Filling in the missing data..........................262 Practical limits on precision in measurement of surface height .......................................262 Stereophotogrammetry of confocal stereo-pair images . 262 Stereoscopic image recording and measurement........262 Reducing the effect of fixed-pattern noise.............263 Confocal pseudoholograms ........................263 Enhanced chromatic aberration methods ...........263 Ubiquity of LCA.................................263 Simple exploitation...............................263 Linear longitudinal chromatic dispersion (LLCD) objectives ....................................263 Color coding with filters...........................264 Some findings .....................................264 Osteoclastic resorption pits.........................264 Local, relative microrelief in tooth wear studies ........265 CHAPTER 16: FLUOROPHORES FOR CONFOCAL MICROSCOPY Roger Y. Tsien and Alan Waggoner Introduction: Photophysics and photochemistry.......267 Photophysical problems related to high-intensity excitation ......................................267 Singlet-state saturation ............................267 Triplet-state saturation ............................268 Contaminating background signals ..................268 Photodestruction of fluorophores and biological specimens......................................269 Dependency on intensity or its time integral? ..........272 Protective agents.................................272 Strategies for signal optimization in the face of photobleaching .................................273 Light-collection efficiency .........................273 Spatial resolution.................................273 Contents xix Fluorophore concentration .........................273 Choice of fluorophore.............................274 Fluorescent labels for antibodies, other proteins, and DNA probes....................................274 Phycobiliproteins ................................274 Green fluorescent protein..........................274 Fluorescent indicators for dynamic intracellular parameters.....................................275 Membrane potentials .............................275 Ion concentrations................................275 Future developments?..............................277 CHAPTER 17: IMAGE CONTRAST IN CONFOCAL LIGHT MICROSCOPY P. C. Cheng and A. Kriete Introduction ......................................281 Sources of contrast ................................281 Confocal microscopy in the backscattered light mode .. 282 Signal formation.................................282 Backscattered light contrast on stained specimens ......284 Image contrast as the result of Rayleigh scattering and the Raman effect ..............................285 Reflection contrast in nonbiological specimens ........285 Backscatter contrast on living specimens .............285 Image contrast as the result of interference............285 The effect of overlying structures ...................285 Absorption contrast...............................288 Transmitted confocal image ........................290 Confocal microscopy in epifluorescence mode........293 Negative contrast when imaging a crystallite ..........293 Effects of specimen optical heterogeneity.............294 Artifacts in fluorescent CLSM......................298 Countermeasures.................................299 Mounting medium selection........................299 Artifacts........................................301 Background level and ghost image from the transmission illuminator ...................................302 Contrast, image quality, and 3D visualization..........304 Summary.........................................308 CHAPTER 18: GUIDING PRINCIPLES OF SPECIMEN PRESERVATION FOR CONFOCAL FLUORESCENCE MICROSCOPY Robert Bacallao, Kianush Kiai, and Lynn Jesaitis Introduction ......................................311 Characteristics of fixatives..........................312 Glutaraldehyde ..................................312 Formaldehyde...................................313 Fixation staining and mounting methods..............313 Glutaraldehyde fixation ...........................313 pH-shift/formaldehyde fixation .....................314 Immunofluorescence staining ......................314 Mounting the specimen ...........................315 Critical evaluation of LM fixation and mounting methods .......................................315 The use of the cell height to evaluate the fixation method 316 Use of cell height to evaluate mounting media......... 316 Well-defined structures can be used to evaluate fixation methods..................................... 317 Comparison of in vi vo-Iabeled cell organelles with immunolabeled cell organelles................... 317 General notes .................................... 318 Labeling samples with two or more probes ........... 319 Triple labeling .................................. 320 Preparation of tissue specimens .................... 320 Refractive index mismatch ........................ 320 Screening antibodies on glutaraldehyde-fixed specimens 320 Future directions ................................ 322 Conclusion....................................... 322 Summary......................................... 323 CHAPTER 19: CONFOCAL MICROSCOPY OF LIVING CELLS M. Terasaki and M. E. Dailey Introduction...................................... 327 General considerations for confocal microscopy of living cells..................................... 327 Maintenance of living cells ........................ 327 Fluorescent probes............................... 328 Reducing photodynamic damage ................... 328 Internet........................................ 330 A convenient test specimen........................ 330 Summary of published work........................ 330 Studies using ion indicator dyes .................... 330 Studies using membrane labeling dyes............... 331 Studies using mRNA probes ....................... 334 Studies using fluorescent analog dyes................ 334 Studies using membrane potential probes............. 334 Studies using dextrans ............................ 335 Studies using calcein AM ......................... 335 Studies using interference reflection................. 335 Tissue studies................................... 335 Specific example I: Dynamics of the endoplasmic reticulum in sea urchin eggs ..................... 335 Changes in ER .................................. 337 Sensitivity to light damage ........................ 338 Specific example II: Axon and dendrite growth in brain slices.......................................... 338 Preparation of CNS tissue slices .................... 339 Staining........................................ 339 Maintaining tissue health on the microscope stage ..... 339 Imaging methods ................................ 340 Imaging deep within tissue ........................ 340 Keeping cells in focus ............................ 341 Handling the data................................ 342 Results ........................................ 343 Conclusions .................................... 343 Future directions.................................. 344 xx Contents CHAPTER 20: LENS ABERRATIONS IN CONFOCAL FLUORESCENCE MICROSCOPY Stefan W. Hell and Ernst H. K. Stelzer Introduction...................................... 347 The situation.................................... 347 Theory........................................... 348 Results of theoretical calculations................... 350 Experiments ...................................... 351 Other considerations .............................. 351 Dry objectives.................................. 351 Refractive index, wavelength and temperature......... 352 Conclusion....................................... 352 Consequences................................... 352 Practical strategies to reduce refractive index mismatch . 353 CHAPTER 21: REAL-TIME STEREO (3D) CONFOCAL MICROSCOPY G. J. Brakenhoff and K. Visscher Introduction...................................... 355 Fundamental constraints........................... 355 General considerations............................ 355 Total fluorescence emission limitations .............. 356 Fluorescent emission flux limitations................ 356 Fundamental limitations, conclusions................ 357 Scanning and image collection techniques ........... 357 Parallel/nonparallel image collection, optical probe size . 357 Point/sequential confocal techniques ................ 358 Direct-field parallel confocal imaging, real-time stereo imaging ..................................... 358 Detectors for real-time confocal imaging............. 361 Discussion ....................................... 361 Summary ........................................ 361 CHAPTER 22: SIGNAL-TO-NOISE IN CONFOCAL MICROSCOPES Colin J. R. Sheppard, Xiaosong Gan, Min Gu, and Maitreyee Roy Introduction...................................... 363 Sources of noise .................................. 363 Shot noise and quantum efficiency.................. 363 Information content .............................. 364 Background noise ............................... 364 Signal level in confocal microscopes................. 365 Signal-to-noise ratio for confocal microscopes........ 366 QE, Nl, and stain level............................ 366 N2 and detectability.............................. 366 Designs of confocal microscope .................... 367 Sampling......................................... 367 Comparative performance of fluorescence microscopes 368 Bleaching limited performance..................... 368 Saturation-limited performance..................... 369 Effects of scanning speed ......................... 369 Three-dimensional imaging........................ 370 Information content of an image.................... 370 Summary ........................................ 370 CHAPTER 23: COMPARISON OF WIDE-FIELD/DECONVOLUTION AND CONFOCAL MICROSCOPY FOR 3D IMAGING Peter J. Shaw Introduction ......................................373 The point spread function: imaging as a convolution ... 373 Deconvolution ....................................376 Nearest neighbor algorithm ........................377 Computing......................................377 Fluorochromes and light sources ....................378 Practical differences ...............................378 Convenience ....................................378 Temporal resolution ..............................378 Sparse sections ..................................379 Integration of fluorescence intensity .................379 Resolution, sensitivity, and noise ....................379 Fluorescence excitation............................379 Fluorescent light detection .........................379 Out-of-focus light ................................380 Model specimens.................................381 Practical comparisons..............................382 Conclusion .......................................384 Summary .........................................385 CHAPTER 24: LIGHT MICROSCOPIC IMAGES RECONSTRUCTED BY MAXIMUM LIKELIHOOD DECONVOLUTION Timothy J. Holmes, Santosh Bhattacharyya, Joshua A. Cooper, David Hanzel, Vijaykumar Krishnamurthi, Wen-chieh Lin, Badrinath Roysam, Donald H. Szarowski, and James N. Turner Introduction ......................................389 Overview.......................................389 Purpose of deblurring .............................389 Main advantages and present limitations..............390 Principles.........................................390 Simplified data collection model ....................390 Maximum likelihood general principles ..............391 General flowchart ................................391 Wide-field fluorescence algorithm...................391 Confocal fluorescence algorithm ....................392 Brightfield algorithm .............................393 Data corrections ..................................393 Image reconstructions..............................394 Simulated wide-field fluorescence...................394 Simulated 2D diffraction-limited wide-field fluorescence with blind deconvolution........................395 Wide-field fluorescence, biological sample............395 Confocal fluorescence, biological samples ............396 Brightfield, biological sample ......................398 Future directions ..................................398 Summary of main points............................400 Contents xxi CHAPTER 25: DIRECT VIEW CONFOCAL IMAGING SYSTEMS USING A SLIT APERTURE W. B. Amos andJ. G. White Introduction ......................................403 Resolution and noise...............................403 Practical implementations ..........................404 Systems with moving slit apertures ..................404 Systems with stationary slit apertures ................405 Performance of stationary-slit systems ...............405 Light sources for slit scanners .......................410 Line generation optics..............................411 Comparison of suitable detectors for slit scanners .....411 Film...........................................413 Electronic image detection.........................413 Extended capabilities with a motorized focus drive .... 413 Extended focus imaging...........................413 Stereo imaging ..................................413 XZ imaging .....................................414 Real-time Z-imaging..............................414 Applications for which slit-scanning confocal systems could offer particular advantages .................414 High-speed epifluorescence........................414 High-sensitivity confocal fluorescence ...............414 CHAPTER 26: TWO NEW HIGH-RESOLUTION CONFOCAL FLUORESCENCE MICROSCOPIES (4PI, THETA) WITH ONE- AND TWO-PHOTON EXCITATION Steffen Lindek, Ernst H. K. Stelzer, and Stefan W. Hell Introduction ......................................417 Principles.........................................417 PSF calculations...................................418 4Pi(a)-confocal fluorescence microscopy.............419 4Pi(b)-confocal scattered light microscopy 420 4Pi(c)-confocal scattered light microscopy............421 Methods to further improve resolution ...............422 Two-photon absorption ...........................422 Fluorescence with a large Stokes shift................423 4Pi(C)-confocal microscopy with complementary interference ..................................424 The phase problem ................................425 Conclusion .......................................426 Appendix: Confocal Theta fluorescence microscopy ... 426 4Pi(a)-confocal Theta microscopy...................427 4Pi(a)-confocal Theta two-photon fluorescence microscopy...................................HZ Experimental setup...............................427 Conclusion .....................................429 CHAPTER 27: OPTICAL CONSIDERATIONS AT ULTRAVIOLET WAVELENGTHS IN CONFOCAL MICROSCOPY A. Christyne Bliton and James D. Lechleiter Introduction ....... Advantages of UV 431 431 UV-confocal microscope design considerations....... 432 UV light sources ................................ 432 UV radiation hazards............................. 432 Detectors....................................... 434 Absorption and dispersion......................... 434 Reflection and antireflection coatings................ 434 Chromatic errors ................................ 435 Immersion medium errors ......................... 435 Chromatic correction techniques.................... 436 Monochromatic aberrations at UV wavelengths ....... 436 Custom optics development........................ 437 Mechanical considerations ........................ 437 Available UV options: Objectives and microscopes .... 437 Features of commercial UV-confocal microscopes .... 438 Future developments and alternative techniques ....... 442 Summary......................................... 443 CHAPTER 28: TWO-PHOTON MOLECULAR EXCITATION IN LASER-SCANNING MICROSCOPY Winfried Denk, David W. Piston, and Watt W. Webb Introduction...................................... 445 Physical principles of two-photon excitation and their implications for image formation ................. 445 Physics of two-photon excitation ................... 445 Optical pulse length.............................. 447 Detection ...................................... 449 Resolution ..................................... 449 Photodamage: Heating and bleaching................ 450 Instrumentation................................... 450 Lasers and the choice of excitation wavelengths ....... 451 Detection ...................................... 452 Optical aberrations............................... 452 Control of laser power and nonmechanical scanning .... 453 Chromophores (fluorophores and caged compounds) .. 453 Two-photon absorption cross sections ............... 453 Fluorescence emission and uncaging ................ 454 Cell viability during imaging........................ 454 Applications of two-photon excitation............... 455 Calcium imaging ................................ 455 Photoactivation (uncaging) ........................ 455 Discussion and outlook ............................ 457 CHAPTER 29: VIDEO-RATE CONFOCAL MICROSCOPY Roger Y. Tsien and Brian J. Bacskai Why try to reach video-rate? ....................... 459 Advantages and disadvantages of Nipkow disks, slit scans, and point laser scans...................... 459 Horizontal scanning at 15 kHz.................... 460 Diffraction-grating-based methods .................. 460 Rotating polygon mirrors.......................... 461 Translating concave mirrors ....................... 461 Resonant galvanometers .......................... 461 Construction of a video-rate, UV-visible laser scanning confocal microscopy prototype .................. 462 Optical layout................................... 462 xxii Contents Sensing the resonant galvanometer position........... 464 Signal acquisition and rectification.................. 465 Video-rate image storage.......................... 465 Image processor hardware and software.............. 466 Commercial resonant-galvanometer-based video-rate confocal microscopy: Nikon Corporation RCM-8000..................................... 467 Performance tests................................. 467 Resolution ..................................... 467 Live cell Ca2+ and cAMP ......................... 470 Fixed specimens................................. 473 A commercial AOD-based confocal microscope for video and higher rates: Noran Instruments Odyssey 473 Conclusions and prospects for the future............. 477 Summary ........................................ 477 CHAPTER 30: CONFOCAL MICROSCOPY WITH TRANSMITTED LIGHT A. E. Dixon and Carol Cogswell Introduction...................................... 479 Instrumentation for transmission confocal ............ 479 Dissector confocal ............................... 479 Double-pass transmission confocal.................. 479 University of Sydney high-resolution confocal transmission system............................. 480 Investigations of confocal versus conventional transmission brightfield and DIC................. 480 Deflection of the focused image spot ................ 482 University of Waterloo confocal transmission system .. 484 Optical scanning mechanisms in transmission microscopy .................................. 486 Summary ........................................ 487 CHAPTER 31: FLUORESCENCE LIFETIME IMAGING, A NEW TOOL IN CONFOCAL MICROSCOPY A. Draaijer, R. Sanders, and H. C. Gerritsen Introduction...................................... 491 Fluorescence, lifetime, and quantum efficiency........ 491 Fluorescence lifetime spectroscopy: Bulk samples ..... 491 Fluorescent lifetime microspectroscopy: Applications .. 492 Fluorescence lifetime imaging methods .............. 494 Introduction .................................... 494 Lifetime sensing in the frequency domain ............ 494 Fluorescence lifetime sensing in the time domain ...... 496 Applications.................................... 499 Probes for fluorescence lifetime microscopy .......... 500 Summary ........................................ 501 CHAPTER 32: IMAGING IMMUNOGOLD LABELS WITH CONFOCAL MICROSCOPY Carol J. Cogswell Introduction...................................... 507 Scattering of light by small particles................. 507 Effect of wavelength on scattering...................508 Effect of particle size on scattering intensity...........508 Imaging small particles with optical microscopes......508 An experimental confocal microscope for imaging immunogold....................................509 Discussion ........................................512 Summary .........................................512 CHAPTER 33: FIBEROPTICS IN CONFOCAL MICROSCOPY P. M. Delaney and M. R. Harris Introduction ......................................515 Fundamentals of fiberoptics.........................515 Gradient index optical fibers .......................516 Modes in optical fibers ............................516 Major applications of optical fibers in confocal microscopy...................................517 Coupling of laser light into optical fibers .............517 The optical fiber as a source ........................518 Single-mode fiber output ..........................518 Projection of fiber output ..........................518 Multimode optical fiber as a laser source..............518 Multimode fiber laser delivery for confocal microscopy .519 The optic fiber as a pinhole .........................520 Practical implementations of fibers in confocal instruments ....................................520 As a source .....................................520 As a detector ....................................520 Separate source and detector fibers ..................520 Same fiber for source and detection..................520 Future directions ..................................521 Miniaturization ..................................521 Lenses that are optically integrated with the fiber tip .... 522 In-fiber passive and active components...............522 Fused biconical taper couplers ......................522 Summary .........................................523 CHAPTER 34: COMPARISON OF VARIOUS OPTICAL SECTIONING METHODS Charles J. Koester Introduction: The scanning slit confocal microscope ... 525 Nonconfocal optical sectioning......................525 Confocal optical sectioning .........................526 Scanning mirror/slit microscope.....................527 Optical sectioning experimental and theoretical.......529 Scanning mirror/slit system ........................529 Confocal pinhole system...........................531 Comparison of slit and pinhole systems...............531 A possible modification of pinhole confocal systems ... 532 Summary comparison of slit and pinhole confocal systems ........................................533 Slit system......................................533 Single pinhole confocal systems ....................533 Resolution vs. detectability .........................533 Contents xxiii CHAPTER 35: MASS STORAGE AND HARD COPY Guy Cox Introduction ......................................535 Mass storage......................................535 Data compression ................................535 Removable storage media..........................537 Hard copy........................................540 Monitors .......................................541 Photographic systems.............................541 Digital printers ..................................542 Hard copy conclusions ............................547 Summary .........................................547 Bulk storage ....................................547 Hard copy ......................................548 CHAPTER 36: TUTORIAL ON PRACTICAL CONFOCAL MICROSCOPY AND USE OF THE CONFOCAL TEST SPECIMEN Victoria Centonze and James Pawley Introduction ......................................549 Getting started...................................549 Bleaching—The only thing that really matters .........549 Getting a confocal image...........................551 No signal.......................................552 Low signal......................................552 Simultaneous detection of BSL and fluorescence.......553 New controls .....................................553 Photon efficiency ................................553 Pinhole size.....................................553 The importance of pixel size .......................556 Use of test specimen...............................558 Why use a test specimen?..........................558 Description of the test specimen ....................558 Mounting a test pattern for fluorescence imaging.......559 Using the test specimen ...........................559 The diatom: A natural 3D test specimen ..............560 Reasons for poor performance ......................563 Sampling problems .............................. 563 Optical problems ................................ 563 Statistical considerations in confocal microscopy ...... 565 Imaging depth .................................. 566 Saturation ...................................... 567 Summary......................................... 567 Appendix: Normal Kohler illumination............... 569 CHAPTER 37: BIBLIOGRAPHY OF CONFOCAL MICROSCOPES Robert H. Webb Books and review articles .......................... 571 Historical interest................................. 571 Theory (mostly)................................... 572 Technical ........................................ 573 Applications...................................... 575 Variants on the main theme ........................ 575 Fiber optic CSMs.................................. 576 Profilometry...................................... 577 Display .......................................... 577 APPENDIX I: OPTICAL UNITS W.B. Amos Radial units ...................................... 579 Axial units ....................................... 579 APPENDIX 2: LIGHT PATHS OF CURRENT COMMERCIAL CONFOCAL LIGHT MICROSCOPES FOR BIOLOGY James Pawley Introduction . INDEX 581 599
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spelling	Handbook of biological confocal microscopy ed. by James B. Pawley 2. ed. New York [u.a.] Plenum Press 1995 XXIII, 632 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Literaturangaben Confocale microscopie gtt Confocal microscopy Microscopy, Confocal Biologie (DE-588)4006851-1 gnd rswk-swf Mikroskopie (DE-588)4039238-7 gnd rswk-swf Biowissenschaften (DE-588)4129772-6 gnd rswk-swf Konfokale Mikroskopie (DE-588)4336446-9 gnd rswk-swf (DE-588)1071861417 Konferenzschrift 1989 San Antonio Tex. gnd-content Mikroskopie (DE-588)4039238-7 s Biologie (DE-588)4006851-1 s DE-604 Konfokale Mikroskopie (DE-588)4336446-9 s Biowissenschaften (DE-588)4129772-6 s Pawley, James B. Sonstige oth HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006873656&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Handbook of biological confocal microscopy Confocale microscopie gtt Confocal microscopy Microscopy, Confocal Biologie (DE-588)4006851-1 gnd Mikroskopie (DE-588)4039238-7 gnd Biowissenschaften (DE-588)4129772-6 gnd Konfokale Mikroskopie (DE-588)4336446-9 gnd
subject_GND	(DE-588)4006851-1 (DE-588)4039238-7 (DE-588)4129772-6 (DE-588)4336446-9 (DE-588)1071861417
title	Handbook of biological confocal microscopy
title_auth	Handbook of biological confocal microscopy
title_exact_search	Handbook of biological confocal microscopy
title_full	Handbook of biological confocal microscopy ed. by James B. Pawley
title_fullStr	Handbook of biological confocal microscopy ed. by James B. Pawley
title_full_unstemmed	Handbook of biological confocal microscopy ed. by James B. Pawley
title_short	Handbook of biological confocal microscopy
title_sort	handbook of biological confocal microscopy
topic	Confocale microscopie gtt Confocal microscopy Microscopy, Confocal Biologie (DE-588)4006851-1 gnd Mikroskopie (DE-588)4039238-7 gnd Biowissenschaften (DE-588)4129772-6 gnd Konfokale Mikroskopie (DE-588)4336446-9 gnd
topic_facet	Confocale microscopie Confocal microscopy Microscopy, Confocal Biologie Mikroskopie Biowissenschaften Konfokale Mikroskopie Konferenzschrift 1989 San Antonio Tex.
url	http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006873656&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT pawleyjamesb handbookofbiologicalconfocalmicroscopy

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