Fundamentals of Light Microscopy and Electronic Imaging

DOUGLAS B. MURPHY supervises core facilities in microscopy and histology at the new HHMI Janelia Farm Research Campus in Ashburn, Virginia. An Adjunct Professor of Cell Biology at Johns Hopkins School of Medicine in Baltimore, Maryland, Dr. Murphy helped establish the School of Medicine Microscope Facility there, which he supervised until 2006. MICHAEL W. DAVIDSON is an assistant scholar/scientist affiliated with the National High Magnetic Field Laboratory and the Department of Biological Science at Florida State University where he is involved in developing educational websites. His digital images and photomicrographs have graced the covers of over 2,000 publications.

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

Object–Image 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

Abbé’s 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 λ-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 (γ) 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