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Tables of Contents for Video Microscopy
Chapter/Section Title
Page #
Page Count
List of Tables
xix
2
Color Plates
xxi
CHAPTER 1 Why Video?
1
12
1.1. Pre-1940: The Invention of TV
1
3
1.2. Earlier Applications of Video to Microscopy
4
2
1.3. Where We Are Today
6
3
1.4. A Few General Remarks
9
4
CHAPTER 2 Microscope Image Formation
13
106
2.1. Introduction: Essential Optical Train of the Light Microscope
13
2
2.2. Imaging Components in the Light Microscope: Magnification
15
7
2.3. Principles of Koehler Illumination
22
4
2.4. Image Resolution and Wave Optics
26
23
2.4.1. The Diffraction Pattern
26
1
2.4.2. Image Resolution and the Diffraction Pattern
27
5
2.4.3. Image Resolution and Spatial Frequency
32
5
2.4.4. Contrast and Modulation Transfer Functions, Test Targets
37
7
2.4.5. Three-Dimensional Diffraction Pattern, Point Spread Function, and Out-of-Focus Image
44
4
2.4.6. Depth of Field
48
1
2.5. Performance Characteristics of Microscope Optics
49
16
2.5.1. Designation of Objective Lenses
50
2
2.5.2. Coverslip Correction
52
3
2.5.3. Tube Lengths and Tube Lenses for Which Microscope Objectives Are Corrected
55
1
2.5.4. Working Distance, Parfocal Distance
56
1
2.5.5. Field Size, Distortion
57
1
2.5.6. Design of Modern Microscope Objectives
58
1
2.5.7. Oculars
59
2
2.5.8. Abberations, Modified Diffraction Patterns, and Point Spread Functions
61
4
2.6. Generation of Image Contrast
65
35
2.6.1. Bright Field, Anaxial Illumination
66
1
2.6.2. Reflection Contrast, Epi-Illumination
67
3
2.6.3. Dark Field
70
1
2.6.4. Phase Contrast, Modulation Contrast, Single-Sideband Edge Enhancement (SSEE)
71
5
2.6.5. Polarizing
76
10
2.6.6. Interference
86
3
2.6.7. Differential Interference Contrast (DIC)
89
2
2.6.8. Fluorescence, Ratio Imaging
91
6
2.6.9. Luminescence
97
2
2.6.10. Holomicrography
99
1
2.7. Scanning Microscopy
100
12
2.7.1. Field Scanning
100
1
2.7.2. Confocal Microscopy
101
4
2.7.3. Two-Photon Excitation
105
2
2.7.4. Proximity Scanning
107
2
2.7.5. Aperture Scanning
109
1
2.7.6. Video Microscopy
110
2
2.8. Optical Sectioning, Stereoscopy, and 3-D Imaging
112
1
2.9. Image Reduction and Optical Manipulation of the Specimen with the Light Microscope
113
6
2.9.1. Microbeam Irradiation, Caged Compounds
115
1
2.9.2. Laser Trapping
116
3
CHAPTER 3 Practical Aspects of Microscopy
119
44
3.1. Adjusting the Microscope for Koehler Illumination
119
6
3.1.1. Alignment and Focus of the Illuminator
119
1
3.1.2. Focusing the Condenser; Alignment of the Illuminator Continued
120
1
3.1.3. Centering the Condenser
121
1
3.1.4. Alignment of Microscopes with a Built-in Illuminator
122
1
3.1.5. Adjustment of the Field Diaphragm
122
1
3.1.6. Adjustment of the Condenser Iris
123
2
3.2. Light Sources and Microscope Image Brightness
125
17
3.2.1. Brightness of Illumination
127
3
3.2.2. Lasers
130
6
3.2.3. Increasing the Signal from the Specimen
136
1
3.2.4. Light-Gathering Power of the Objective Lens
137
1
3.2.5. Image Brightness and Magnification
137
2
3.2.6. Light Transmission through the Microscope
139
3
3.3. Choice and Care of Lenses for Video Microscopy
142
15
3.3.1. Objective Lenses, Mechanical Standards
142
1
3.3.2. Oculars for Video Microscopy
143
4
3.3.3. Collector and Codenser Lenses
147
2
3.3.4. Inspecting and Cleaning the Optics and Video Camera Faceplate
149
8
3.4. The Microscope Stand
157
6
CHAPTER 4 Physiological Characteristics of the Eye
163
26
4.1. Introduction
163
1
4.2. Structure, Refraction, and Accommodation of the Human Eye
163
2
4.3. Visual Sensitivity and Adaptation
165
5
4.4. Resolution, Visual Acuity
170
3
4.5. Contrast Discrimination and the Modulation Transfer Function
173
5
4.6. Flicker
178
2
4.7. Color Vision
180
4
4.8. Stereoscopy
184
2
4.9. Additional Remarks
186
3
CHAPTER 5 Video Signal Fundamentals
189
44
5.1. The Video Image and Signal
189
2
5.2. How Video Works
191
6
5.2.1. Generation of the Video Signal
191
1
5.2.2. Sequential Scanning
192
1
5.2.3. H and V Scans
192
2
5.2.4. H and V Deflections
194
1
5.2.5. Blanking
195
1
5.2.6. Standard Scanning Rates
195
1
5.2.7. Interlacing
196
1
5.2.8. Active Picture Area
196
1
5.2.9. The Picture Signal
196
1
5.3. Timing Pulses and the Composite Video Signal
197
17
5.3.1. Sync Pulses and Composite Video
197
2
5.3.2. Conventions and Standards for CCTV
199
2
5.3.3. Sync Pulses during V Blanking
201
2
5.3.4. Other Interlace and Timing Conventions
203
3
5.3.5. High-Definition Television
206
1
5.3.6. Visualizing the Waveform, Pulse-Cross Display
207
7
5.4. Image Resolution and the Video Signal
214
11
5.4.1. Video Resolution Defined
214
1
5.4.2. Vertical Resolution
215
2
5.4.3. Horizontal Resolution
217
4
5.4.4. Modulation Transfer Function in Video
221
2
5.4.5. Signal-to-Noise Ratio in Video
223
2
5.5. Synchronizing the Video Equipment
225
8
5.5.1. Compatibilities of Scan Rates and Sync Pulses
225
3
5.5.2. Signal Termination
228
1
5.5.3. Sync Stripper
228
3
5.5.4. Distribution Amplifier
231
2
CHAPTER 6 Detection of Light and Monochrome Vidicon Cameras
233
40
6.1. Photomultiplier Tubes
233
5
6.1.1. Responsivity
233
1
6.1.2. Photocathode Properties
234
1
6.1.3. Electron Multiplication
234
1
6.1.4. Dark Current
235
1
6.1.5. Signal-to-Noise (S/N) Ration
236
2
6.2. Imaging: From Photomultiplier Tubes to Video
238
7
6.2.1. Area Detection
238
1
6.2.2. Image Dissector Tube
238
3
6.2.3. The Modern Vidicon
241
4
6.3. Characterizing Video Sensors
245
20
6.3.1. Sensitivity, Responsivity, and Wavelength Dependence
246
1
6.3.2. Light Transfer Characteristics
247
2
6.3.3. Gray Scale
249
1
6.3.4. Shading
250
1
6.3.5. Dark Current and Electrical Noise
251
3
6.3.6. MTF, Resolution, and Contrast
254
4
6.3.7. Dynamic Range
258
1
6.3.8. Geometrical Distortion
258
1
6.3.9. Lag
259
3
6.3.10. Blooming
262
2
6.3.11. Burn, Sticking
264
1
6.3.12. Blemishes
264
1
6.4. Performance Comparisons
265
8
6.4.1. Sensitivity
265
1
6.4.2. Contrast
265
1
6.4.3. Dynamic Range
266
1
6.4.4. Signal-Processing Features
267
1
6.4.5. Scan Rates, Interlace, and Clock Stability
268
1
6.4.6. Scan Direction and Image Size
268
1
6.4.7. External Sync Capability
268
1
6.4.8. Other Considerations
269
4
CHAPTER 7 Solid-State Detectors and Cameras
273
54
7.1. Simple Solid-State Detectors
273
3
7.1.1. Photodiodes
273
2
7.1.2. Avalanche and Hybrid Photodiodes
275
1
7.2. Spatial Discrimination with Solid-State Detector Arrays
276
2
7.2.1. Split Detectors
276
1
7.2.2. MOS Photodetector
276
2
7.3. CCD Fundamentals
278
16
7.3.1. Charge Storage
278
1
7.3.2. Charge Transfer
279
4
7.3.3. Readout
283
1
7.3.4. CCD Geometry
283
2
7.3.5. Spectral Sensitivity
285
1
7.3.6. Gamma
285
1
7.3.7. Shading and Charge-Transfer Efficiency
286
1
7.3.8. S/N Ratio
287
2
7.3.9. Modulation Transfer Function
289
1
7.3.10. Dynamic Range
290
2
7.3.11. Geometrical Distortion
292
1
7.3.12. Lag
292
1
7.3.13. Burning and Blooming
292
1
7.3.14. Electronic Shuttering and On-Chip Integration
292
2
7.4. Other Video-Rate Solid-State Sensors and Cameras
294
11
7.4.1. The HAD CCD
294
2
7.4.2. HyperHAD CCD
296
1
7.4.3. The Charge Injection Device (CID)
297
4
7.4.4. Solid-State Cameras with Built-in Digitizers
301
3
7.4.5. CCD Cameras for HDTV
304
1
7.4.6. Time-Delay Integration (TDI)
305
1
7.5. Slow-Scan CCD Cameras
305
16
7.5.1. Background
305
1
7.5.2. Some Distinctions
306
1
7.5.3. Sensor Geometry
306
3
7.5.4. Spectral Sensitivity
309
1
7.5.5. Light Transfer Characteristics
310
2
7.5.6. Shading
312
1
7.5.7. Modulation Transfer Function
312
1
7.5.8. Noise in Slow-Scan Cameras
313
1
7.5.9. S/N Ratio, Dynamic Range, and Minimum Detectable Signal
314
2
7.5.10. Camera Design Considerations
316
2
7.5.11. Subarray Sampling
318
1
7.5.12. Binning
319
1
7.5.13. Slow-Scan versus Video-Rate Cameras
320
1
7.6. High-Speed Imaging with Solid-State Detectors
321
6
7.6.1. General Considerations
321
1
7.6.2. Multiple-Output CCDs
321
1
7.6.3. Burst Imaging with Slow-Scan Detectors
321
1
7.6.4. High-Speed Imaging with MOS Arrays
322
1
7.6.5. Streak Cameras
323
4
CHAPTER 8 Low-Light-Level Cameras
327
40
8.1. The Development of Low-Light-Level Video-Rate Cameras
327
9
8.1.1. Early Low-Light-Level Video Cameras
327
2
8.1.2. Silicon-Intensifier Target Camera
329
1
8.1.3. First-Generation Image Intensifiers
330
2
8.1.4. Intensifier Silicon-Intensifier Target Camera
332
1
8.1.5. Three-Stage Intensified Cameras
333
1
8.1.6. S/N Ratio
333
3
8.2. Newer-Generation Image Intensifiers
336
9
8.2.1. Second-Generation Image Intensifiers
336
7
8.2.2. Proximity-Focused Image Intensifiers
343
1
8.2.3. Third- and Fourth-Generation Image Intensifiers
344
1
8.3. Coupling Intensifiers to Video-Rate Cameras and Slow-Scan CCDs
345
4
8.3.1. Relay Lens Coupling
345
2
8.3.2. Fiber-Optic Coupling
347
1
8.3.3. Lens Coupling versus Fiber Taper
347
1
8.3.4. The Intensified CCD (ICCD) Camera
348
1
8.4. Photon-Counting Detectors
349
4
8.4.1. Position-Sensitive Detectors
349
3
8.4.2. Electron-Bombarded CCD (EBCCD)
352
1
8.5. Low-Light-Level Imaging Requirements in Microscopy
353
14
8.5.1. Light Levels in Microscopy
353
1
8.5.2. Spatial versus Temporal Resolution
354
3
8.5.3. Photometric Linearity and Dynamic Range
357
1
8.5.4. Geometrical Distortion
358
1
8.5.5. Speed of Response
359
4
8.5.6. Shading
363
1
8.5.7. ICCD versus Slow-Scan CCD in Low-Light-Level Microscopy
363
3
8.5.8. Comparing Low-Light-Level Cameras in the Laboratory
366
1
CHAPTER 9 Color Video Signal and Color Cameras
367
16
9.1. Color Fundamentals
367
3
9.1.1. Color Standards
368
1
9.1.2. Graphical Representation of Color
369
1
9.2. Color Video Encoding
370
6
9.3. Color Video Cameras
376
5
9.3.1. Color Camera Performance
377
3
9.3.2. Integrating Color Cameras
380
1
9.4. Some Considerations Regarding Color Signals
381
2
CHAPTER 10 Video Monitors, Projectors, and Printers
383
58
10.1. Some General Considerations
383
1
10.2. Monochrome CRT Video Monitors
384
22
10.2.1. The Picture Tube and Basic Monitor Circuitry
384
1
10.2.2. Picture Size
385
3
10.2.3. Resolution of the Monitor
388
1
10.2.4. Gamma, Contrast, and Picture Brightness
389
2
10.2.5. Types of Phosphors, Hue, and Persistence
391
1
10.2.6. Geometrical Distortions
391
6
10.2.7. Shading, Streaking, Ringing, and Other Picture Defects
397
1
10.2.8. Critical Adjustment and Testing of the Monitor
398
2
10.2.9. Monitors with Special Features
400
6
10.3. Color CRT Video Monitors
406
3
10.4. Solid-State and Plasma Display Monitors--Monochrome and Color
409
7
10.4.1. Liquid-Crystal Displays
409
5
10.4.2. Plasma Displays
414
2
10.5. Video Projectors
416
4
10.5.1. CRT Video Projectors
417
1
10.5.2. Light Valve Projectors
418
2
10.5.3. Projectors Based on Spatial Light Modulators
420
1
10.6. Video Printers and Production of Video Micrographs
420
9
10.6.1. Video Printers
422
2
10.6.2. Photographing the Monitor
424
3
10.6.3. Scan-Line Removal
427
2
10.6.4. Dedicated Video Film Recorders
429
1
10.7. Optical Copying with Video
429
5
10.8. Stereo Video Displays
434
7
CHAPTER 11 Video Recorders
441
68
11.1. Introduction
441
1
11.2. Video Tape Recording
441
16
11.2.1. How VTRs Work
441
5
11.2.2. The Scanner
446
1
11.2.3. Tape Path and Transport
447
2
11.2.4. Servo Controls
449
1
11.2.5. Head Configurations
450
1
11.2.6. Time Encoding
450
3
11.2.7. Frequency Response and Signal Processing in Magnetic Tape Recording
453
4
11.3. Videotape Formats--Monochrome and Color
457
10
11.3.1. Three-Quarter-Inch U format
458
1
11.3.2. Half-Inch Formats
458
9
11.4. Characterization of VTRs for Video Microscopy
467
5
11.4.1. Resolution
468
1
11.4.2. Contrast and Picture Definition
469
1
11.4.3. Still-Frame and Variable-Speed Display
469
1
11.4.4. Editing Capability
469
1
11.4.5. Portability, RF Conversion
470
1
11.4.6. Compatibility
470
1
11.4.7. Ease of Use, Convenience Features
471
1
11.4.8. Reliability, Availability of Service
471
1
11.5. Time-Lapse VTRs, Time-Base Errors, High-Speed VTRs
472
4
11.5.1. Time-Lapse VTRs
472
1
11.5.2. Commercially Available Time-Lapse VTRs
473
1
11.5.3. Some Practical Considerations
473
1
11.5.4. Time-Base Error
474
2
11.5.5. High-Speed Recording
476
1
11.6. Care and Maintenance of the VCR
476
6
11.6.1. General Precautions
477
1
11.6.2. Playback
477
2
11.6.3. Recording Operation
479
2
11.6.4. Troubleshooting and Repair
481
1
11.6.5. Head Cleaning and Replacement
481
1
11.6.6. When Not in Use
482
1
11.7. Choice and Care of the Videotape
482
2
11.7.1. Choice of Tape
483
1
11.7.2. Storage of Recorded Tape
483
1
11.8. Copying and Editing onto Videotape
484
6
11.8.1. The Equipment
484
2
11.8.2. Selecting and Organizing the Scenes
486
1
11.8.3. Title Making
487
1
11.8.4. The Mechanics of Editing
487
3
11.9. Special Effects
490
2
11.10. Video Disk Recorders
492
11
11.10.1. Magnetic Disk Recorders
494
1
11.10.2. Optical Disk Recorders and Players
494
6
11.10.3. Magneto-Optical Disk Recording
500
2
11.10.4. Holographic Image Storage
502
1
11.11. Digital Video Recording
503
6
11.11.1. Image Storage in Memory
503
1
11.11.2. Compression of Digitized Images
504
1
11.11.3. Image Storage on Hard Disks
504
1
11.11.4. CD-ROM Players
505
1
11.11.5. Rewritable Optical Disk Storage of Video Images
506
1
11.11.6. Magnetic or Optical Digital Disk versus VTR Recording
506
3
CHAPTER 12 Digital Image Processing
509
50
12.1. Introduction
509
1
12.2. Digital Image Acquisition
510
9
12.2.1. Digitizing the Output of an Analog Video Camera
510
1
12.2.2. Required Amplitude Resolution
510
3
12.2.3. Digital Camera Output
513
1
12.2.4. Sampling Frequency Requirements
513
1
12.2.5. Aliasing
514
1
12.2.6. Pixel Geometry
515
1
12.2.7. Control of A/D Parameters
515
2
12.2.8. A/D Noise
517
2
12.3. Image Processor Architecture
519
1
12.4. Digital Signal Processing
520
10
12.4.1. Digital Adjustment of Offset and Gain
520
4
12.4.2. Image Integration
524
4
12.4.3. Image Averaging
528
2
12.5. Image Analysis--Measurement of Intensity
530
9
12.5.1. Line Intensity Scan
530
1
12.5.2. Gray-Level Histogram
531
2
12.5.3. Clipping
533
1
12.5.4. Histogram-Based Operations
534
1
12.5.5. Subtraction of a Background Image
535
1
12.5.6. Ratio Imaging
536
3
12.6. Image Analysis--Morphometric Operations
539
3
12.6.1. The Binary Image
539
1
12.6.2. Dilation and Erosion
539
1
12.6.3. Segmentation
540
1
12.6.4. Additional Binary Operations
541
1
12.7. Image Processing--Convolutions, Filters, Transforms
542
11
12.7.1. Convolution Kernels
544
1
12.7.2. Spatial Averaging
545
1
12.7.3. Sharpening Filters
546
1
12.7.4. Unsharp Masking
547
1
12.7.5. Derivative Filters
547
1
12.7.6. Fourier Transforms
547
2
12.7.7. Deconvolution
549
3
12.7.8. Warping and Geometrical Transforms
552
1
12.8. Display of Digitized Images
553
6
12.8.1. Output LUTs
553
2
12.8.2. Pan, Scroll, and Zoom
555
1
12.8.3. Display of Multiple Images
556
3
CHAPTER 13 System Integration
559
56
13.1. Initial Adjustments and Connections in a Basic System
559
13
13.1.1. The Minimum
559
1
13.1.2. Adjustment and Hookup of the Video Camera and Monitor
559
5
13.1.3. Coupling the Video Camera and the Microscope
564
1
13.1.4. Wait! Read Before Turning the Microscope Lamp On
564
2
13.1.5. Adjustments of the Monitor
566
1
13.1.6. Termination
567
1
13.1.7. Aligning the Video Camera with the Microscope
568
4
13.2. Component Selection
572
3
13.2.1. Transmitted-Light Microscopy
572
1
13.2.2. Polarized-Light, Dark-Field, Reflection Contrast, and DIC Microscopy and Related Techniques
573
1
13.2.3. Fluorescence Microscopy of Fixed Preparations
573
1
13.2.4. Fluorescence Microscopy of Living Cells, Ratio Imaging, and Related Techniques
574
1
13.3. Making Connections
575
9
13.3.1. Wiring Diagrams
575
4
13.3.2. Physical Arrangements of Wiring
579
1
13.3.3. Component Matching
580
2
13.3.4. Computer Interfacing
582
2
13.4. Reducing Noise
584
11
13.4.1. Degradation of the Video Signal by Optical Noise
584
7
13.4.2. Degradation of the Video Signal by Electronic Noise
591
4
13.5. Environmental Considerations
595
8
13.5.1. Equipment Environment
595
1
13.5.2. Specimen Temperature Control
596
2
13.5.3. Observation and Perfusion Chambers
598
5
13.6. The Limits of Video Microscopy
603
12
13.6.1. Multiview Microscopy
604
1
13.6.2. Flow and Permeability Measurements
605
3
13.6.3. High-Speed Microscopy
608
1
13.6.4. Optical Traps and Force Measurements
609
2
13.6.5. Single-Fluorophore Detection
611
3
13.6.6. Concluding Remarks
614
1
Postscript
615
4
References
619
16
Glossary
635
70
List of Manufacturers
705
6
Index
711