Handbook of biological confocal microscopy
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
New York [u.a.]
Plenum Press
1995
|
| Ausgabe: | 2. ed. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV010328579 | ||
| 003 | DE-604 | ||
| 005 | 19970415 | ||
| 007 | t| | ||
| 008 | 950804s1995 xx ad|| |||| 00||| eng d | ||
| 020 | |a 0306448262 |9 0-306-44826-2 | ||
| 035 | |a (OCoLC)31710400 | ||
| 035 | |a (DE-599)BVBBV010328579 | ||
| 040 | |a DE-604 |b ger |e rakddb | ||
| 041 | 0 | |a eng | |
| 049 | |a DE-355 |a DE-20 |a DE-91G |a DE-M49 |a DE-19 |a DE-703 |a DE-11 |a DE-188 |a DE-578 | ||
| 050 | 0 | |a QH224 | |
| 082 | 0 | |a 578/.45 |2 20 | |
| 084 | |a WC 2900 |0 (DE-625)148075: |2 rvk | ||
| 084 | |a WC 2905 |0 (DE-625)148076: |2 rvk | ||
| 084 | |a BIO 040f |2 stub | ||
| 084 | |a PHY 131f |2 stub | ||
| 245 | 1 | 0 | |a Handbook of biological confocal microscopy |c ed. by James B. Pawley |
| 250 | |a 2. ed. | ||
| 264 | 1 | |a New York [u.a.] |b Plenum Press |c 1995 | |
| 300 | |a XXIII, 632 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Literaturangaben | ||
| 650 | 7 | |a Confocale microscopie |2 gtt | |
| 650 | 4 | |a Confocal microscopy | |
| 650 | 4 | |a Microscopy, Confocal | |
| 650 | 0 | 7 | |a Biologie |0 (DE-588)4006851-1 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Mikroskopie |0 (DE-588)4039238-7 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Biowissenschaften |0 (DE-588)4129772-6 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Konfokale Mikroskopie |0 (DE-588)4336446-9 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)1071861417 |a Konferenzschrift |y 1989 |z San Antonio Tex. |2 gnd-content | |
| 689 | 0 | 0 | |a Mikroskopie |0 (DE-588)4039238-7 |D s |
| 689 | 0 | 1 | |a Biologie |0 (DE-588)4006851-1 |D s |
| 689 | 0 | |5 DE-604 | |
| 689 | 1 | 0 | |a Konfokale Mikroskopie |0 (DE-588)4336446-9 |D s |
| 689 | 1 | 1 | |a Biowissenschaften |0 (DE-588)4129772-6 |D s |
| 689 | 1 | |5 DE-604 | |
| 700 | 1 | |a Pawley, James B. |e Sonstige |4 oth | |
| 856 | 4 | 2 | |m HBZ Datenaustausch |q application/pdf |u 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 |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-006873656 | |
Datensatz im Suchindex
| DE-19_call_number | 1705/E 2 Paw 15755=LS Soft Condensed Matter 1705/E 2 Paw 14452=LS Photonics and Optoelectronics 1705/WC 2905 P339(2) 1900/WC 2905 P339(2) |
|---|---|
| DE-19_location | 95 29 |
| DE-BY-UBM_katkey | 1457872 |
| DE-BY-UBM_local_call_number | 1705/E 2 Paw 14452=Lehrstuhl Feldmann 1705/E 2 Paw 15755=Lehrstuhl Rädler |
| DE-BY-UBM_local_notation | LS Rädler LS Feldmann |
| DE-BY-UBM_media_number | 99992369507 41612016370017 41601303990013 99992267542 |
| DE-BY-UBR_call_number | 00/WC 2900 P339(2) |
| DE-BY-UBR_katkey | 2179383 |
| DE-BY-UBR_location | 00 |
| DE-BY-UBR_media_number | 069007336500 |
| _version_ | 1823051110435258368 |
| 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
|
| any_adam_object | 1 |
| building | Verbundindex |
| bvnumber | BV010328579 |
| callnumber-first | Q - Science |
| callnumber-label | QH224 |
| callnumber-raw | QH224 |
| callnumber-search | QH224 |
| callnumber-sort | QH 3224 |
| callnumber-subject | QH - Natural History and Biology |
| classification_rvk | WC 2900 WC 2905 |
| classification_tum | BIO 040f PHY 131f |
| ctrlnum | (OCoLC)31710400 (DE-599)BVBBV010328579 |
| dewey-full | 578/.45 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 578 - Natural history of organisms |
| dewey-raw | 578/.45 |
| dewey-search | 578/.45 |
| dewey-sort | 3578 245 |
| dewey-tens | 570 - Biology |
| discipline | Physik Biologie |
| edition | 2. ed. |
| format | Book |
| fullrecord | <?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>02028nam a2200529 c 4500</leader><controlfield tag="001">BV010328579</controlfield><controlfield tag="003">DE-604</controlfield><controlfield tag="005">19970415 </controlfield><controlfield tag="007">t|</controlfield><controlfield tag="008">950804s1995 xx ad|| |||| 00||| eng d</controlfield><datafield tag="020" ind1=" " ind2=" "><subfield code="a">0306448262</subfield><subfield code="9">0-306-44826-2</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(OCoLC)31710400</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)BVBBV010328579</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-604</subfield><subfield code="b">ger</subfield><subfield code="e">rakddb</subfield></datafield><datafield tag="041" ind1="0" ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="049" ind1=" " ind2=" "><subfield code="a">DE-355</subfield><subfield code="a">DE-20</subfield><subfield code="a">DE-91G</subfield><subfield code="a">DE-M49</subfield><subfield code="a">DE-19</subfield><subfield code="a">DE-703</subfield><subfield code="a">DE-11</subfield><subfield code="a">DE-188</subfield><subfield code="a">DE-578</subfield></datafield><datafield tag="050" ind1=" " ind2="0"><subfield code="a">QH224</subfield></datafield><datafield tag="082" ind1="0" ind2=" "><subfield code="a">578/.45</subfield><subfield code="2">20</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">WC 2900</subfield><subfield code="0">(DE-625)148075:</subfield><subfield code="2">rvk</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">WC 2905</subfield><subfield code="0">(DE-625)148076:</subfield><subfield code="2">rvk</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">BIO 040f</subfield><subfield code="2">stub</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">PHY 131f</subfield><subfield code="2">stub</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Handbook of biological confocal microscopy</subfield><subfield code="c">ed. by James B. Pawley</subfield></datafield><datafield tag="250" ind1=" " ind2=" "><subfield code="a">2. ed.</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="a">New York [u.a.]</subfield><subfield code="b">Plenum Press</subfield><subfield code="c">1995</subfield></datafield><datafield tag="300" ind1=" " ind2=" "><subfield code="a">XXIII, 632 S.</subfield><subfield code="b">Ill., graph. Darst.</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="b">n</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="b">nc</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="500" ind1=" " ind2=" "><subfield code="a">Literaturangaben</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">Confocale microscopie</subfield><subfield code="2">gtt</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Confocal microscopy</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Microscopy, Confocal</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Biologie</subfield><subfield code="0">(DE-588)4006851-1</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Mikroskopie</subfield><subfield code="0">(DE-588)4039238-7</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Biowissenschaften</subfield><subfield code="0">(DE-588)4129772-6</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Konfokale Mikroskopie</subfield><subfield code="0">(DE-588)4336446-9</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="655" ind1=" " ind2="7"><subfield code="0">(DE-588)1071861417</subfield><subfield code="a">Konferenzschrift</subfield><subfield code="y">1989</subfield><subfield code="z">San Antonio Tex.</subfield><subfield code="2">gnd-content</subfield></datafield><datafield tag="689" ind1="0" ind2="0"><subfield code="a">Mikroskopie</subfield><subfield code="0">(DE-588)4039238-7</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="0" ind2="1"><subfield code="a">Biologie</subfield><subfield code="0">(DE-588)4006851-1</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="0" ind2=" "><subfield code="5">DE-604</subfield></datafield><datafield tag="689" ind1="1" ind2="0"><subfield code="a">Konfokale Mikroskopie</subfield><subfield code="0">(DE-588)4336446-9</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="1" ind2="1"><subfield code="a">Biowissenschaften</subfield><subfield code="0">(DE-588)4129772-6</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="1" ind2=" "><subfield code="5">DE-604</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Pawley, James B.</subfield><subfield code="e">Sonstige</subfield><subfield code="4">oth</subfield></datafield><datafield tag="856" ind1="4" ind2="2"><subfield code="m">HBZ Datenaustausch</subfield><subfield code="q">application/pdf</subfield><subfield code="u">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</subfield><subfield code="3">Inhaltsverzeichnis</subfield></datafield><datafield tag="943" ind1="1" ind2=" "><subfield code="a">oai:aleph.bib-bvb.de:BVB01-006873656</subfield></datafield></record></collection> |
| genre | (DE-588)1071861417 Konferenzschrift 1989 San Antonio Tex. gnd-content |
| genre_facet | Konferenzschrift 1989 San Antonio Tex. |
| id | DE-604.BV010328579 |
| illustrated | Illustrated |
| indexdate | 2025-02-03T16:34:53Z |
| institution | BVB |
| isbn | 0306448262 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-006873656 |
| oclc_num | 31710400 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-20 DE-91G DE-BY-TUM DE-M49 DE-BY-TUM DE-19 DE-BY-UBM DE-703 DE-11 DE-188 DE-578 |
| owner_facet | DE-355 DE-BY-UBR DE-20 DE-91G DE-BY-TUM DE-M49 DE-BY-TUM DE-19 DE-BY-UBM DE-703 DE-11 DE-188 DE-578 |
| physical | XXIII, 632 S. Ill., graph. Darst. |
| publishDate | 1995 |
| publishDateSearch | 1995 |
| publishDateSort | 1995 |
| publisher | Plenum Press |
| record_format | marc |
| 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 |