Douglas B. Murphy9780471253914, 0-471-25391-X
Table of contents :
Front Cover……Page 1
Contents……Page 6
Preface……Page 12
4.1……Page 14
4.2……Page 15
9.1……Page 16
11.1……Page 17
Optical Components of the Light Microscope……Page 18
Note: Inverted Microscope Designs……Page 20
Aperture and Image Planes in a Focused, Adjusted Microscope……Page 21
Koehler Illumination……Page 23
Note: Summary of Steps for Koehler Illumination……Page 24
Precautions for Handling Optical Equipment……Page 28
Exercise: Calibration of Magnification……Page 29
Light As a Probe of Matter……Page 32
Light as Particles and Waves……Page 35
The Quality of Light……Page 37
Properties of Light Perceived by the Eye……Page 38
Physical Basis for Visual Perception and Color……Page 39
Positive and Negative Colors……Page 41
Exercise: Complementary colors……Page 43
Illuminators and Their Spectra……Page 46
Demonstration: Spectra of Common Light Sources……Page 50
Illuminator Alignment and Bulb Replacement……Page 51
Demonstration: Aligning a 100 W Mercury Arc Lamp in an Epi-Illuminator……Page 52
“First on-Last Off”: Essential Rule for Arc Lamp Power Supplies……Page 53
Filters for Adjusting the Intensity and Wavelength of Illumination……Page 54
Neutral Density Filters……Page 55
Interference Filters……Page 56
Effects of Light on Living Cells……Page 58
Image Formation by a Simple Lens……Page 60
Note: Real and Virtual Images……Page 62
Object-Image Math……Page 63
The Principal Aberrations of Lenses……Page 67
Designs and Specifications of Objective Lenses……Page 70
Markings on the Barrel of a Lens……Page 71
Image Brightness……Page 72
Oculars……Page 73
Microscope Slides and Coverslips……Page 74
The Care and Cleaning of Optics……Page 75
Exercise: Constructing and Testing an Optical Bench Microscope……Page 76
Defining Diffraction and Interference……Page 78
The Diffraction Image of a Point Source of Light……Page 81
Demonstration: Viewing the Airy Disk with a Pinhole Aperture……Page 83
Constancy of Optical Path Length Between the Object and the Image……Page 85
Effect of Aperture Angle on Diffraction Spot Size……Page 86
Diffraction by a Grating and Calculation of Its Line Spacing, D……Page 88
Demonstration: The Diffraction Grating……Page 92
Abbe’s Theory for Image Formation in the Microscope……Page 94
Diffraction Pattern Formation in the Back Aperture of the Objective Lens……Page 97
Demonstration: Observing the Diffraction Image in the Back Focal Plane of a Lens…….Page 98
Preservation of Coherence: an Essential Requirement for Image Formation……Page 99
Exercise: Diffraction by Microscope Specimens……Page 101
Numerical Aperture……Page 102
Spatial Resolution……Page 104
Depth of Field and Depth of Focus……Page 107
Optimizing the Microscope Image: A Compromise Between Spatial Resolution and Contrast……Page 108
Exercise: Resolution of Striae in Diatoms……Page 110
Phase Contrast Microscopy……Page 114
The Behavior of Waves from Phase Objects in Bright-Field Microscopy……Page 116
The Optical Design of the Phase Contrast Microscope……Page 120
Interpretating the Phase Contrast Image……Page 123
Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry……Page 127
Theory and Optics……Page 129
Image Interpretation……Page 132
Exercise: Dark-Field Microscopy……Page 133
The Generation of Polarized Light……Page 134
Demonstration: Producing Polarized Light with a Polaroid Filter……Page 136
Vectorial Analysis of Polarized Light Using a Dichroic Filter……Page 138
Double Refraction in Crystals……Page 141
Demonstration: Double Refraction by a Calcite Crystal……Page 143
Kinds of Birefringence……Page 144
Propagation of O and E Wavefronts in a Birefringent Crystal……Page 145
Birefringence in Biological Specimens……Page 147
Generation of Elliptically Polarized Light by Birefringent Specimens……Page 148
Overview……Page 152
Optics of the Polarizing Microscope……Page 153
Adjusting the Polarizing Microscope……Page 155
Principles of Action of Retardation Plates and Three Popular Compensators……Page 156
Demonstration: Making a Plate from a Piece of Cellophane……Page 160
Exercise: Determination of Molecular Organization in Biological Structures Using a Full Wave Plate Compensator……Page 165
The DIC Optical System……Page 170
DIC Equipment and Optics……Page 172
The DIC Prism……Page 174
Demonstration: The Action of a Wollaston Prism in Polarized Light……Page 175
Formation of the DIC Image……Page 176
Interference Between O and E Wavefronts and the Application of Bias Retardation……Page 177
Alignment of DIC Components……Page 178
Image Interpretation……Page 183
The Use of Compensators in DIC Microscopy……Page 184
Modulation Contrast Microscopy……Page 185
Contrast Methods Using Oblique Illumination……Page 186
Alignment of the Modulation Contrast Microscope……Page 189
Exercise: DIC Microscopy……Page 190
Overview……Page 194
Applications of Fluorescence Microscopy……Page 195
Physical Basis of Fluorescence……Page 196
Properties of Fluorescent Dyes……Page 199
Demonstration: Fluorescence of Chlorophyll and Fluorescein……Page 200
Autofluorescence of Endogenous Molecules……Page 202
Arrangement of Filters and the Epi-illuminator in the Fluorescence Microscope……Page 206
Objective Lenses and Spatial Resolution in Fluorescence Microscopy……Page 211
Causes of High-Fluorescence Background……Page 213
The Problem of Bleed-Through with Multiply Stained Specimens……Page 214
Examining Fluorescent Molecules in Living Cells……Page 215
Exercise: Fluorescence Microscopy of Living Tissue Culture Cells……Page 216
Overview……Page 222
The Optical Principle of Confocal Imaging……Page 225
Demonstration: Isolation of Focal Plane Signals with a Confocal Pinhole……Page 228
Advantages of CLSM Over Wide-Field Fluorescence Systems……Page 230
Criteria Defining Image Quality and the Performance of an Electronic Imaging System……Page 232
Electronic Adjustments and Considerations for Confocal Fluorescence Imaging……Page 234
Photobleaching……Page 240
General Procedure for Acquiring a Confocal Image……Page 241
Two-Photon and Multi-Photon Laser Scanning Microscopy……Page 243
Confocal Imaging with a Spinning Nipkow Disk……Page 246
Exercise: Effect of Confocal Variables on Image Quality……Page 247
Applications and Specimens Suitable for Video……Page 250
Configuration of a Video Camera System……Page 251
Types of Video Cameras……Page 253
Electronic Camera Controls……Page 255
Demonstration: Procedure for Adjusting the Light Intensity of the Video Camera and TV Monitor……Page 258
Video Enhancement of Image Contrast……Page 259
Criteria Used to Define Video Imaging Performance……Page 262
Aliasing……Page 265
Digital Image Processors……Page 266
Image Intensifiers……Page 267
VCRs……Page 268
Systems Analysis of a Video Imaging System……Page 269
Daisy Chaining a Number of Signal-Handling Devices……Page 271
Exercise: Contrast Adjustment and Time-Lapse Recording with a Video Camera……Page 272
Overview……Page 276
The Charge-Coupled Device (CCD Imager)……Page 277
CCD Architectures……Page 284
Note: Interline CCDs for Biomedical Imaging……Page 285
Camera Acquisition Parameters Affecting CCD Readout and Image Quality……Page 286
Imaging Performance of a CCD Detector……Page 288
Requirements and Demands of Digital CCD Imaging……Page 293
Color Cameras……Page 294
Points to Consider When Choosing a Camera……Page 295
Exercise: Evaluating the Performance of a CCD Camera……Page 296
Overview……Page 300
Preliminaries: Image Display and Data Types……Page 301
Histogram Adjustment……Page 302
Adjusting Gamma (y) to Create Exponential LUTs……Page 304
Flat-Field Correction……Page 306
Image Processing with Filters……Page 309
Signal-to-Noise Ratio……Page 316
Exercise: Flat-Field Correction and Determination of S/N Ratio……Page 322
Image Processing: One Variable Out of Many Affecting the Appearance of the Microscope Image……Page 324
Record Keeping During Image Acquisition and Processing……Page 326
Note: Guidelines for Image Acquisition and Processing……Page 327
Use of Color in Prints and Image Displays……Page 329
Colocalization of Two Signals Using Pseudocolor……Page 330
A Checklist for Evaluating Image Quality……Page 332
Answer Key to Problem Sets……Page 334
Materials for Demonstrations and Exercises……Page 338
Sources of Materials for Demonstrations and Exercises……Page 346
Glossary……Page 348
References……Page 374
Index……Page 378
Back Cover……Page 386
LinkToy : )~……Page 0
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