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Tables of Contents for Computer Operation for Microscope Photometry
Chapter/Section Title
Page #
Page Count
Chapter 1. Introduction
1
28
1.1 Purpose of this Book
1
1
1.2 Power Supply
2
1
1.2.1 Stabilization
2
1
1.2.2 Arc Lamps
3
1
1.3 Illuminator
3
2
1.3.1 Adjustment
3
1
1.3.2 Assembly
4
1
1.4 Kohler Illumination
5
3
1.4.1 Lamp Size
5
1
1.4.2 Adjustments
5
2
1.4.3 Testing
7
1
1.4.4 Problems
8
1
1.5 Resolution
8
2
1.5.1 Numerical Aperture
9
1
1.5.2 Comparison with Electron Microscope
9
1
1.6 Microscope Objectives
10
9
1.6.1 Achromats
11
1
1.6.2 Cleaning
11
1
1.6.3 Enhanced Achromats
11
1
1.6.4 Apochromats
12
1
1.6.5 Fluorite Objectives
12
1
1.6.6 Polarized Light Objectives
13
1
1.6.7 Epi-Objectives
13
2
1.6.8 Phase Contrast
15
2
1.6.9 Differential Interference-Contrast
17
2
1.7 PC Interfacing
19
6
1.7.1 GPIB (HP-IB or IEEE 488)
19
2
1.7.2 RS-232
21
3
1.7.3 VXI
24
1
1.8 Software
25
4
1.8.1 Graphics
25
1
1.8.2 Different Types of Scanning
26
1
1.8.3 Dimensions of Scanning
27
2
Chapter 2. Illuminators
29
6
2.1 Introduction
29
1
2.2 Arc Lamps
29
3
2.2.1 Historical
29
1
2.2.2 Short-Arc Mercury Lamp
30
1
2.2.3 Short-Arc Xenon Lamp
31
1
2.3 Tungsten-Halogen Filament Lamps
32
1
2.4 Emission Spectra
32
1
2.5 Control of Illumination Intensity
33
2
Chapter 3. Shutters and Apertures
35
24
3.1 Introduction
35
1
3.2 Shutter and Aperture Locations
36
3
3.2.1 Illumination Shutter
37
1
3.2.2 Photometric Field Aperture
37
1
3.2.3 Ocular Shutter
37
1
3.2.4 Photometer Shutter
38
1
3.2.5 Photometric Measuring Aperture
38
1
3.2.6 Secondary Illuminator Shutter
38
1
3.2.7 Protective Shutter
39
1
3.3 Shutter-Photometer Interactions
39
10
3.3.1 Hardware
40
1
3.3.2 Software
41
3
3.3.3 PMT Response
44
3
3.3.4 Comparison of Two Shutters
47
1
3.3.5 Minimum Exposure Protocol
47
2
3.4 Shutter Software for Normal Operations
49
7
3.4.1 Power-Up and Power-Down
49
1
3.4.2 Measuring Position
49
1
3.4.3 Viewing Position
50
1
3.4.4 Visible Light Viewing Position for Ultraviolet Fluorometry
50
1
3.4.5 Standardization of the Photometer for Fluorometry
50
1
3.4.6 Dark-Field
50
1
3.4.7 Dark-Field plus Ambient
50
1
3.4.8 Negative Shutter Test
51
1
3.4.9 Positive Shutter Test
51
1
3.4.10 Diagnostics
51
1
3.4.11 Software
51
5
3.5 Importance of Timing
56
3
Chapter 4. Photometry
59
26
4.1 Introduction
59
1
4.2 Analog-to-Digital Conversion
59
1
4.2.1 Dual-Slope Method
59
1
4.2.2 Successive Approximation Method
60
1
4.2.3 Nyquist Theorem
60
1
4.3 Photoresistive Detectors
60
2
4.3.1 Spectral Response
61
1
4.4 Photovoltaic Detectors
62
1
4.4.1 Solar Cell
62
1
4.5 Photomultipliers
63
17
4.5.1 Hardware
63
1
4.5.2 Software
64
16
4.6 Monitoring Performance
80
5
4.6.1 Different Controllers
80
1
4.6.2 Transient Events
81
1
4.6.3 Amplifier Gain
82
1
4.6.4 Good and Bad PMT Settings
82
3
Chapter 5. Monochromators and Spectrophotometry
85
16
5.1 Introduction
85
1
5.2 Step-and-Measure
85
3
5.2.1 Data Structure
85
2
5.2.2 Scanner Hierarchy
87
1
5.2.3 Spectral Scanning Algorithm
87
1
5.3 Monochromators
88
11
5.3.1 Software Options
88
1
5.3.2 Prism Monochromator
89
1
5.3.3 Grating Monochromator
90
2
5.3.4 Stray-Light Filters
92
1
5.3.5 Monochromator Location
93
1
5.3.6 Continuous Interference Filter
94
1
5.3.7 Software
94
5
5.4 Distributional Error
99
2
Chapter 6. Mechanical Stages and Scanning
101
12
6.1 Introduction
101
1
6.2 Stepper Motors
102
5
6.2.1 Operational Problems
103
1
6.2.2 Software
104
3
6.3 Histochemical Mapping
107
5
6.3.1 Hardware
108
1
6.3.2 Software
108
2
6.3.3 Results
110
2
6.4 Switching Optical Fibers
112
1
6.5 Tilting Stages
112
1
Chapter 7. Polarized Light
113
24
7.1 Introduction
113
5
7.1.1 Principal Axes
113
1
7.1.2 Types of Birefringence
114
1
7.1.3 Retardation
114
1
7.1.4 Interference Colors
115
1
7.1.5 Types of Compensators
115
1
7.1.6 de Senarmont Compensator Algorithm
116
2
7.1.7 Brace-Kohler Compensator Algorithm
118
1
7.2 Knob Rotation Actuator
118
2
7.2.1 Operational Features
119
1
7.3 Continuous Measurement of Birefringence
120
3
7.3.1 Hardware
120
1
7.3.2 Software
121
1
7.3.3 Results and Practical Problems
121
2
7.4 Tilting Stage and Polarization of Reflected Light
123
11
7.4.1 Hardware
124
1
7.4.2 Theory
124
2
7.4.3 Illumination and Tilting Restraints
126
1
7.4.4 Extinction Coefficient
127
1
7.4.5 Intrinsic Anisotropy
127
1
7.4.6 Vertical 0(0) vs. Lateral 45(0)
127
2
7.4.7 Alignment and Depth of Focus
129
1
7.4.8 Surface Irregularities
129
1
7.4.9 Spectrophotometry of Tilted Standards
130
1
7.4.10 Spectrophotometry of Tissue Surface Reflectance
131
1
7.4.11 Spectrophotometry of Diffuse Reflectance
132
1
7.4.12 Polarimetry of Surface Reflectance
132
2
7.4.13 Iridescence
134
1
7.5 Why Is the Polarized Light Microscope Seldom Used by Biologists?
134
3
Chapter 8. Fluorescence
137
16
8.1 Introduction
137
1
8.2 Excitation vs. Emission
138
1
8.3 Dia-Excitation
139
1
8.3.1 Ultraviolet Safety
140
1
8.4 Epi-Excitation
140
2
8.5 Importance of Collagen Autofluorescence
142
1
8.6 Fluorometry Software
143
6
8.6.1 Relative Spectral Fluorescence Intensity
143
1
8.6.2 Spectrofluorometry
144
2
8.6.3 Comparison of Methods at High Light Intensity
146
1
8.6.4 Comparison of Methods at Low Light Intensity
147
1
8.6.5 High vs. Low Light Intensity for Standardization
148
1
8.7 Fluorescence Blank and Pseudofluorescence
149
4
Chapter 9. Video
153
12
9.1 Introduction
153
1
9.2 Pixels and Gray Levels
154
1
9.3 Video Cameras
154
2
9.4 Software
156
7
9.4.1 Noise
156
1
9.4.2 Contrast Enhancement
156
3
9.4.3 Neighborhood Averaging Kernels
159
3
9.4.4 Laplacian Kernels
162
1
9.4.5 Erosion and Dilation
162
1
9.5 Summary
163
2
Chapter 10. Photodiode Array Spectrography
165
10
10.1 Introduction
165
1
10.1.1 Advantage of PDA Over Shutter Pulse
165
1
10.2 Mounting the PDA
166
1
10.2.1 PDA Spectrograph
166
1
10.2.2 CAM
167
1
10.3 Retrospective Standardization
167
2
10.4 Fluorometry of Collagen and Elastin
169
6
10.4.1 Sources of Variance: Samples vs. Photodiodes
169
3
10.4.2 Software for Rate of Quenching
172
1
10.4.3 Effect of pH and Measuring Direction
172
3
Chapter 11. Liberating the Microscope with Fiberoptics
175
28
11.1 Introduction
175
1
11.2 Optical Fibers
176
4
11.2.1 Step-Index Fibers
176
1
11.2.2 Numerical Aperture
176
1
11.2.3 Monomode Fibers
177
1
11.2.4 Coupling
177
1
11.2.5 Fiber Arrangements
178
1
11.2.6 Internal Reflectance in Tissues
179
1
11.3 Remote Spectrophotometry
180
4
11.3.1 Standardization
181
2
11.3.2 Examples
183
1
11.4 Spatial Scanning
184
3
11.4.1 Monochromatic Scattering with a Laser
185
1
11.4.2 Polychromatic Scattering with a CAM
186
1
11.5 Goniospectrophotometry
187
2
11.5.1 Hardware
188
1
11.5.2 Standardization
189
1
11.6 Remote Fluorometry
189
10
11.6.1 Needle Penetration Fluorometry
190
1
11.6.2 Vignette Window Effect
191
5
11.6.3 Spectral Window Effect
196
1
11.6.4 Internal Reflections
197
1
11.6.5 Hypodermic Needle Angle
198
1
11.6.6 Widespread Occurrence of Aperture Effects
198
1
11.7 Coherent Fiberoptic Light Guide for Remote Imaging
199
1
11.8 Polarized Light
199
4
11.8.1 Transmittance
200
1
11.8.2 Back-Scattering
201
2
Chapter 12. Programming the Sample Environment
203
16
12.1 Introduction
203
1
12.2 Regulating pH
203
9
12.2.1 Hardware
204
2
12.2.2 Software
206
4
12.2.3 Example
210
2
12.3 Refractive Index Control
212
1
12.4 Temperature Control
212
3
12.4.1 Temperature Sensor
213
1
12.4.2 Thermal Stage
213
2
12.4.3 Control Loops
215
1
12.5 Planning the Experimental Protocol
215
4
References
219
14
Index
233