Light and Video Microscopy (eBook)

Front Cover 1
Light and Video Microscopy 4
Copyright Page 5
Contents 8
Preface 12
Chapter 1. The Relation between the Object and the Image 14
Luminous and Nonluminous Objects 14
Object and Image 14
Theories of Vision 15
Light Travels in Straight Lines 18
Images Formed in a Camera Obscura: Geometric Considerations 18
Where Does Light Come From? 21
How Can the Amount of Light Be Measured? 23
Chapter 2. The Geometric Relationship between Object and Image 24
Reflection by a Plane Mirror 24
Reflection by a Curved Mirror 26
Reflection from Various Sources 29
Images Formed by Refraction at a Plane Surface 29
Images Formed by Refraction at a Curved Surface 33
Fermat's Principle 39
Optical Path Length 41
Lens Aberrations 42
Geometric Optics and Biology 44
Geometric Optics of the Human Eye 44
Web Resources 46
Chapter 3. The Dependence of Image Formation on the Nature of Light 48
Christiaan Huygens and the Invention of the Wave Theory of Light 48
Thomas Young and the Development of the Wave Theory of Light 52
James Clerk Maxwell and the Wave Theory of Light 64
Ernst Abbe and the Relationship of Diffraction to Image Formation 66
Resolving Power and the Limit of Resolution 73
Contrast 76
Web Resource 78
Chapter 4. Bright-Field Microscopy 80
Components of the Microscope 81
The Optical Paths of the Light Microscope 86
Using the Bright-Field Microscope 89
Depth of Field 92
Out-of-Focus Contrast 93
Uses of Bright-Field Microscopy 93
Care and Cleaning of the Light Microscope 93
Web Resources 94
Chapter 5. Photomicrography 96
Setting up the Microscope for Photomicrography 96
Scientific History of Photography 98
General Nature of the Photographic Process 100
The Resolution of the Film 103
Exposure and Composition 104
The Similarities between Film and the Retina 106
Web Resources 106
Chapter 6. Methods of Generating Contrast 108
Dark-Field Microscopy 108
Rheinberg Illumination 110
Oblique Illumination 111
Phase-Contrast Microscopy 112
Oblique Illumination Reconsidered 121
Annular Illumination 122
Chapter 7. Polarization Microscopy 124
What Is Polarized Light? 124
Use an Analyzer to Test for Polarized Light 126
Production of Polarized Light 127
Influencing Light 132
Design of a Polarizing Microscope 133
What Is the Molecular Basis of Birefringence? 135
Interference of Polarized Light 139
The Origin of Colors in Birefringent Specimens 146
Use of Compensators to Determine the Magnitude and Sign of Birefringence 146
Crystalline versus Form Birefringence 157
Orthoscopic versus Conoscopic Observations 158
Reflected Light Polarization Microscopy 158
Uses of Polarization Microscopy 158
Optical Rotatory (or Rotary) Polarization and Optical Rotatory (or Rotary) Dispersion 160
Web Resources 161
Chapter 8. Interference Microscopy 162
Generation of Interference Colors 162
The Relationship of Interference Microscopy to Phase-Contrast Microscopy 165
Quantitative Interference Microscopy: Determination of the Refractive Index, Mass, Concentration of Dry Matter, Concentration of Water, and Density 167
Source of Errors When Using an Interference Microscope 169
Making a Coherent Reference Beam 170
Double-Beam versus Multiple-Beam Interference 171
Interference Microscopes Based on a Mach-Zehnder Type Interferometer 171
Interference Microscopes Based on Polarized Light 173
The Use of Transmission Interference Microscopy in Biology 177
Reflection-Interference Microscopy 177
Uses of Reflection-Interference Microscopy in Biology 178
Chapter 9. Differential Interference Contrast (DIC) Microscopy 180
Design of a Transmitted Light Differential Interference Contrast Microscope 180
Interpretation of a Transmitted Light Differential Interference Contrast Image 182
Design of a Reflected Light Differential Interference Contrast Microscope 186
Interpretation of a Reflected Light Differential Interference Contrast Image 187
Chapter 10. Amplitude Modulation Contrast Microscopy 188
Hoffman Modulation Contrast Microscopy 189
Reflected Light Hoffman Modulation Contrast Microscopy 192
The Single-Sideband Edge Enhancement Microscope 192
Chapter 11. Fluorescence Microscopy 196
Discovery of Fluorescence 196
Physics of Fluorescence 197
Design of a Fluorescence Microscope 203
Fluorescence Probes 205
Pitfalls and Cures in Fluorescence Microscopy 206
Web Resources 206
Chapter 12. Various Types of Microscopes and Accessories 208
Confocal Microscopes 208
Laser Microbeam Microscope 211
Optical Tweezers 212
Laser Capture Microdissection 212
Laser Doppler Microscope 213
Centrifuge Microscope 213
X-Ray Microscope 214
Infrared Microscope 215
Nuclear Magnetic Resonance Imaging Microscope 215
Stereo Microscopes 215
Scanning Probe Microscopes 216
Acoustic Microscope 217
Horizontal and Traveling Microscopes 218
Microscopes for Children 218
Microscope Accessories 218
Web Resources 219
Chapter 13. Video and Digital Microscopy 220
The Value of Video and Digital Microscopy 220
Video and Digital Cameras: The Optical to Electrical Signal Converters 221
Monitors: Conversion of an Electronic Signal into an Optical Signal 229
Storage of Video and Digital Images 230
Connecting a Video System 231
Web Resources 231
Chapter 14. Image Processing and Analysis 232
Analog Image Processing 232
Digital Image Processing 237
Enhancement Functions of Digital Image Processors 238
Analysis Functions of Digital Image Processors 241
The Ethics of Digital Image Processing 242
Web Resources 243
Chapter 15. Laboratory Exercises 244
Laboratory 1: The Nature of Light and Geometric Optics 244
Laboratory 2: Physical Optics 246
Laboratory 3: The Bright-Field Microscope and Image Formation 247
Laboratory 4: Phase-Contrast Microscopy, Dark-Field Microscopy, Rheinberg Illumination, and Oblique Illumination 249
Laboratory 5: Fluorescence Microscopy 252
Laboratory 6: Polarized Light 253
Laboratory 7: Polarizing Light Microscopy 254
Laboratory 8: Interference Microscopy 254
Laboratory 9: Differential Interference Contrast Microscopy and Hoffman Modulation Contrast Microscopy 255
Laboratory 10: Video and Digital Microscopy and Analog and Digital Image Processing 256
Commercial Sources for Laboratory Equipment and Specimens 258
References 260
Appendix I. A Final Exam 286
Appendix II. A Microscopist's Model of the Photon 290
Index 298
A 298
B 298
C 298
D 299
E 299
F 299
G 299
H 300
I 300
J 300
K 300
L 300
M 300
N 301
O 301
P 301
Q 301
R 301
S 302
T 302
U 302
V 302
W 302
X 302
Y 302
Z 302
Color Plates 304