Light and video microscopy

308 Pages·2014·6.074 MB·English

by  Wayne, Randy

Light and Video Microscopy
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Light and Video Microscopy
Randy Wayne

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD
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This book is dedicated to my brother Scott.
Light and Video Microscopy
v
Copyright © 2009 by Academic Press. Inc. All rights of reproduction in any form reserved.
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Contents
Preface xi 4. Bright-Field Microscopy 67
Components of the Microscope 68
1. The Relation between the Object
The Optical Paths of the Light Microscope 73
and the Image 1
Using the Bright-Field Microscope 76
Luminous and Nonluminous Objects 1 Depth of Field 79
Object and Image 1 Out-of-Focus Contrast 80
Theories of Vision 2 Uses of Bright-Field Microscopy 80
Light Travels in Straight Lines 5 Care and Cleaning of the Light Microscope 80
Images Formed in a Camera Obscura: Geometric Web Resources 81
Considerations 5
Where Does Light Come From? 8 5. Photomicrography 83
How Can the Amount of Light Be Measured? 10
Setting up the Microscope for Photomicrography 83
Scientific History of Photography 85
2. The Geometric Relationship between
General Nature of the Photographic Process 87
Object and Image 11 The Resolution of the Film 90
Exposure and Composition 91
Reflection by a Plane Mirror 11
The Similarities between Film and the Retina 93
Reflection by a Curved Mirror 13
Web Resources 93
Reflection from Various Sources 16
Images Formed by Refraction at a Plane Surface 16
6. Methods of Generating Contrast 95
Images Formed by Refraction at a Curved Surface 20
Fermat’s Principle 26
Dark-Field Microscopy 95
Optical Path Length 28
Rheinberg Illumination 97
Lens Aberrations 29
Oblique Illumination 98
Geometric Optics and Biology 31
Phase-Contrast Microscopy 99
Geometric Optics of the Human Eye 31
Oblique Illumination Reconsidered 108
Web Resources 33 Annular Illumination 109
3. The Dependence of Image Formation 7. Polarization Microscopy 111
on the Nature Of Light 35
W hat Is Polarized Light? 111
C hristiaan Huygens and the Invention of the Use an Analyzer to Test for Polarized Light 113
Wave Theory of Light 35 Production of Polarized Light 114
Thomas Young and the Development of the Wave Influencing Light 119
Theory of Light 39 Design of a Polarizing Microscope 120
James Clerk Maxwell and the Wave Theory of Light 51 What Is the Molecular Basis of Birefringence? 122
Ernst Abbe and the Relationship of Diffraction to Interference of Polarized Light 126
Image Formation 53 The Origin of Colors in Birefringent Specimens 133
Resolving Power and the Limit of Resolution 60 Use of Compensators to Determine the
Contrast 63 Magnitude and Sign of Birefringence 133
Web Resources 65 Crystalline versus Form Birefringence 144
vii
viii Contents
Orthoscopic versus Conoscopic Observations 145 Fluorescence Probes 192
Reflected Light Polarization Microscopy 145 Pitfalls and Cures in Fluorescence Microscopy 193
Uses of Polarization Microscopy 145 Web Resources 193
Optical Rotatory (or Rotary) Polarization and
Optical Rotatory (or Rotary) Dispersion 147 12. Various Types of Microscopes and
Web Resources 148 Accessories 195
Confocal Microscopes 195
8. Interference Microscopy 149
Laser Microbeam Microscope 198
G eneration of Interference Colors 149 Optical Tweezers 199
T he Relationship of Interference Microscopy Laser Capture Microdissection 199
to Phase-Contrast Microscopy 152 Laser Doppler Microscope 200
Quantitative Interference Microscopy: Centrifuge Microscope 200
Determination of the Refractive Index, Mass, X-Ray Microscope 201
Concentration of Dry Matter, Concentration of Infrared Microscope 202
Water, and Density 154 Nuclear Magnetic Resonance Imaging
Source of Errors When Using an Interference Microscope 202
Microscope 156 Stereo Microscopes 202
Making a Coherent Reference Beam 157 Scanning Probe Microscopes 203
Double-Beam versus Multiple-Beam Acoustic Microscope 204
Interference 158 Horizontal and Traveling Microscopes 205
Interference Microscopes Based on a Mach- Microscopes for Children 205
Zehnder Type Interferometer 158 Microscope Accessories 205
Interference Microscopes Based on Polarized Web Resources 206
Light 160
The Use of Transmission Interference 13. Video and Digital Microscopy 207
Microscopy in Biology 164
The Value of Video and Digital Microscopy 207
Reflection-Interference Microscopy 164
Video and Digital Cameras: The Optical to
Uses of Reflection-Interference Microscopy
Electrical Signal Converters 208
in Biology 165
Monitors: Conversion of an Electronic Signal
into an Optical Signal 216
9. Differential Interference Contrast
Storage of Video and Digital Images 217
(DIC) Microscopy 167
Connecting a Video System 218
D esign of a Transmitted Light Differential Web Resources 218
Interference Contrast Microscope 167
Interpretation of a Transmitted Light 14. Image Processing and Analysis 219
Differential Interference Contrast Image 169
Analog Image Processing 219
Design of a Reflected Light Differential
Digital Image Processing 224
Interference Contrast Microscope 173
Enhancement Functions of Digital Image
Interpretation of a Reflected Light Differential
Interference Contrast Image 174 Processors 225
Analysis Functions of Digital Image Processor s 228
The Ethics of Digital Image Processing 229
10. Amplitude Modulation Contrast
Web Resources 230
Microscopy 175
Hoffman Modulation Contrast Microscopy 176 15. Laboratory Exercises 231
Reflected Light Hoffman Modulation Contrast
Laboratory 1: The Nature of Light and
Microscopy 179
Geometric Optics 231
The Single-Sideband Edge Enhancement
Laboratory 2: Physical Optics 233
Microscope 179
Laboratory 3: The Bright-Field Microscope
and Image Formation 234
11. Fluorescence Microscopy 183
Laboratory 4: Phase-Contrast Microscopy,
Discovery of Fluorescence 183 Dark-Field Microscopy, Rheinberg
Physics of Fluorescence 184 Illumination, and Oblique Illumination 236
Design of a Fluorescence Microscope 190 Laboratory 5: Fluorescence Microscopy 239