Preface xi
Acknowledgments xii
1. Fundamentals of Light Microscopy 1
Overview 1
Optical Components of the Light Microscope 1
Aperture and Image Planes in a Focused, Adjusted Microscope 5
Note: Objectives, Eyepieces, and Eyepiece Telescopes 6
Koehler Illumination 9
Adjusting the Microscope for Koehler Illumination 9
Note: Summary of Steps for Koehler Illumination 11
Note: Focusing Oil Immersion Objectives 14
Fixed Tube Length versus Infinity Optical Systems 15
Precautions for Handling Optical Equipment 16
Care and Maintenance of the Microscope 17
Exercise: Calibration of Magnification 17
2. Light and Color 21
Overview 21
Light as a Probe of Matter 21
The Dual Particle- and Wave-Like Nature of Light 25
The Quality of Light 26
Properties of Light Perceived by the Eye 27
Physical Basis for Visual Perception and Color 28
Addition and Subtraction Colors 30
Exercise: Complementary Colors 32
3. Illuminators, Filters, and the Isolation of Specific Wavelengths 35
Overview 35
Illuminators and Their Spectra 35
Illuminator Alignment and Bulb Replacement 41
Demonstration: Spectra of Common Light Sources 41
Demonstration: Aligning a 100-W Mercury Arc Lamp in an Epi-Illuminator 43
Filters for Adjusting the Intensity and Wavelength of Illumination 45
Effects of Light on Living Cells 50
4. Lenses and Geometrical Optics 53
Overview 53
Reflection and Refraction of Light 53
Image Formation by a Simple Lens 56
Note: Real and Virtual Images 57
Rules of Ray Tracing for a Simple Lens 58
ObjectImage Math 58
The Principal Aberrations of Lenses 62
Designs and Specifications of Objectives 65
Condensers 71
Oculars 72
Microscope Slides and Coverslips 73
The Care and Cleaning of Optics 73
Exercise: Constructing and Testing an Optical Bench Microscope 76
5. Diffraction and Interference in Image Formation 79
Overview 79
Diffraction and Interference 80
The Diffraction Image of a Point Source of Light 83
The Constancy of Optical Path Length between Object and Image 85
Demonstration: Viewing the Airy Disk with a Pinhole Aperture 85
Effect of Aperture Angle on Diffraction Spot Size 87
Diffraction by a Grating and Calculation of Its Line Spacing,D 89
Demonstration: The Diffraction Grating 93
Abbés Theory for Image Formation in the Microscope 94
A Diffraction Pattern Is Formed in the Rear Aperture of the Objective 97
Demonstration: Observing the Diffraction Image in the Rear Focal Plane of a Lens 98
Preservation of Coherence: Essential Requirement for Image Formation 99
Exercise: Diffraction by Microscope Specimens 101
6. Diffraction and Spatial Resolution 103
Overview 103
Numerical Aperture 103
Spatial Resolution 105
Depth of Field and Depth of Focus 109
Optimizing the Microscope Image: A Compromise between Spatial Resolution and Contrast 109
Exercise: Resolution of Striae in Diatoms 112
7. Phase Contrast Microscopy and Darkfield Microscopy 115
Overview 115
Phase Contrast Microscopy 115
The Behavior of Waves from Phase Objects in Brightfield Microscopy 119
Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry 128
Darkfield Microscopy 129
Exercise: Darkfield Microscopy 133
8. Properties of Polarized Light 135
Overview 135
The Generation of Polarized Light 135
Demonstration: Producing Polarized Light with a Polaroid Filter 137
Polarization by Reflection and Scattering 139
Vectorial Analysis of Polarized Light Using a Dichroic Filter 139
Double Refraction in Crystals 142
Demonstration: Double Refraction by a Calcite Crystal 144
Kinds of Birefringence 145
Propagation of O and E Wavefronts in a Birefringent Crystal 146
Birefringence in Biological Specimens 148
Generation of Elliptically Polarized Light by Birefringent Specimens 149
9. Polarization Microscopy 153
Overview 153
Optics of the Polarizing Microscope 155
Adjusting the Polarizing Microscope 156
Appearance of Birefringent Objects in Polarized Light 157
Principles of Action of Retardation Plates and Three Popular Compensators 158
Demonstration: Making a-Plate from a Piece of Cellophane 162
Exercise: Determination of Molecular Organization in Biological Structures Using a Full Wave Plate Compensator 167
10. Differential Interference Contrast Microscopy and Modulation Contrast Microscopy 173
Overview 173
The DIC Optical System 173
Demonstration: The Action of a Wollaston Prism in Polarized Light 179
Modulation Contrast Microscopy 190
Exercise: DIC Microscopy 194
11. Fluorescence Microscopy 199
Overview 199
Applications of Fluorescence Microscopy 201
Physical Basis of Fluorescence 202
Properties of Fluorescent Dyes 205
Demonstration: Fluorescence of Chlorophyll and Fluorescein 206
Autofluorescence of Endogenous Molecules 211
Demonstration: Fluorescence of Biological Materials under UV Light 213
Fluorescent Dyes and Proteins in Fluorescence Microscopy 213
Arrangement of Filters and the Epi-Illuminator in the Fluorescence Microscope 218
Objectives and Spatial Resolution in Fluorescence Microscopy 224
Causes of High Fluorescence Background 225
The Problem of Bleedthrough with Multiply Stained Specimens 227
Quenching, Blinking, and Photobleaching 228
Examining Fluorescent Molecules in Living Cells 230
12. Fluorescence Imaging of Dynamic Molecular Processes 233
Overview 233
Modes of Dynamic Fluorescence Imaging 234
Förster Resonance Energy Transfer 236
Applications 244
Fluorescence Recovery after Photobleaching 245
TIRF Microscopy: Excitation by an Evanescent Wave 252
Advanced and Emerging Dynamic Fluoresence Techniques 261
13. Confocal Laser Scanning Microscopy 265
Overview 265
The Optical Principle of Confocal Imaging 267
Demonstration: Isolation of Focal Plane Signals with a Confocal Pinhole 271
Advantages of CLSM over Widefield Fluorescence Systems 273
Criteria Defining Image Quality and the Performance of an Electronic Imaging System 275
Confocal Adjustments and Their Effects on Imaging 277
Photobleaching 286
General Procedure for Acquiring a Confocal Image 286
Performance Check of a Confocal System 288
Fast (Real-Time) Imaging in Confocal Microscopy 288
Spectral Analysis: A Valuable Enhancement for Confocal Imaging 295
Optical Sectioning by Structured Illumination 297
Deconvolution Microscopy 298
Exercise: Effect of Confocal Variables on Image Quality 304
14. Two-photon Excitation Fluorescence Microscopy 307
Overview 307
The Problem of Photon Scattering in Deep Tissue Imaging 308
Two-Photon Excitation Is a Nonlinear Process 309
Localization of Excitation 314
Why Two-Photon Imaging Works 317
Resolution 318
Equipment 319
Three-Photon Excitation 325
Second Harmonic Generation Microscopy 326
15. Superresolution Imaging 331
Overview 331
The RESOLFT Concept 333
Single-Molecule Localization Microscopy 334
Structured Illumination Microscopy 343
Stimulated Emission Depletion (STED) Microscopy: Superresolution by PSF Engineering 349
16. Imaging Living Cells with the Microscope 357
Overview 357
Labeling Strategies for Live-Cell Imaging 358
Control of Illumination 361
Control of Environmental Conditions 365
Optics, Detectors, and Hardware 372
Evaluating Live-Cell Imaging Results 384
Exercise: Fluorescence Microscopy of Living Tissue Culture Cells 384
17. Fundamentals of Digital Imaging 389
Overview 389
The Charge-Coupled Device (CCD Imager) 390
CCD Designs 396
Note: Interline CCD Imagers: The Design of Choice for Biomedical Imaging 398
Back-Thinned Sensors 398
EMCCD Cameras: High Performance Design for Greatest Sensitivity 399
Scientific CMOS: The Next Generation of Scientific Imagers 400
Camera Variables Affecting CCD Readout and Image Quality 401
Six Terms Define Imaging Performance 404
Aliasing 409
Color Cameras 410
Exercise: Evaluating the Performance of a CCD Camera 411
18. Digital Image Processing 415
Overview 415
Preliminaries: Image Display and Data Types 416
Histogram Adjustment 417
Adjusting Gamma () to Create Exponential LUTs 421
Flat-Field Correction 421
Image Processing With Filters 425
Signal-to-Noise Ratio 432
The Use of Color 438
Images as Research Data and Requirements for Scientific Publication 442
Exercise: Flat-Field Correction and Determination of S/N Ratio 448
Appendix A: Answer Key to Exercises 451
Appendix B: Materials for Demonstrations and Exercises 455
Appendix C: Sources of Materials for Demonstrations and Exercises 463
Glossary 465
Microscopy Web Resources 509
Recommended Reading 521
References 523
Index 531
