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 xiAcknowledgments xii1. Fundamentals of Light Microscopy 1Overview 1Optical Components of the Light Microscope 1Aperture and Image Planes in a Focused, Adjusted Microscope 5Note: Objectives, Eyepieces, and Eyepiece Telescopes 6Koehler Illumination 9Adjusting the Microscope for Koehler Illumination 9Note: Summary of Steps for Koehler Illumination 11Note: Focusing Oil Immersion Objectives 14Fixed Tube Length versus Infinity Optical Systems 15Precautions for Handling Optical Equipment 16Care and Maintenance of the Microscope 17Exercise: Calibration of Magnification 172. Light and Color 21Overview 21Light as a Probe of Matter 21The Dual Particle- and Wave-Like Nature of Light 25The Quality of Light 26Properties of Light Perceived by the Eye 27Physical Basis for Visual Perception and Color 28Addition and Subtraction Colors 30Exercise: Complementary Colors 323. Illuminators, Filters, and the Isolation of Specific Wavelengths 35Overview 35Illuminators and Their Spectra 35Illuminator Alignment and Bulb Replacement 41Demonstration: Spectra of Common Light Sources 41Demonstration: Aligning a 100-W Mercury Arc Lamp in an Epi-Illuminator 43Filters for Adjusting the Intensity and Wavelength of Illumination 45Effects of Light on Living Cells 504. Lenses and Geometrical Optics 53Overview 53Reflection and Refraction of Light 53Image Formation by a Simple Lens 56Note: Real and Virtual Images 57Rules of Ray Tracing for a Simple Lens 58Object–Image Math 58The Principal Aberrations of Lenses 62Designs and Specifications of Objectives 65Condensers 71Oculars 72Microscope Slides and Coverslips 73The Care and Cleaning of Optics 73Exercise: Constructing and Testing an Optical Bench Microscope 765. Diffraction and Interference in Image Formation 79Overview 79Diffraction and Interference 80The Diffraction Image of a Point Source of Light 83The Constancy of Optical Path Length between Object and Image 85Demonstration: Viewing the Airy Disk with a Pinhole Aperture 85Effect of Aperture Angle on Diffraction Spot Size 87Diffraction by a Grating and Calculation of Its Line Spacing, D 89Demonstration: The Diffraction Grating 93Abbé’s Theory for Image Formation in the Microscope 94A Diffraction Pattern Is Formed in the Rear Aperture of the Objective 97Demonstration: Observing the Diffraction Image in the Rear Focal Plane of a Lens 98Preservation of Coherence: Essential Requirement for Image Formation 99Exercise: Diffraction by Microscope Specimens 1016. Diffraction and Spatial Resolution 103Overview 103Numerical Aperture 103Spatial Resolution 105Depth of Field and Depth of Focus 109Optimizing the Microscope Image: A Compromise between Spatial Resolution and Contrast 109Exercise: Resolution of Striae in Diatoms 1127. Phase Contrast Microscopy and Darkfield Microscopy 115Overview 115Phase Contrast Microscopy 115The Behavior of Waves from Phase Objects in Brightfield Microscopy 119Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry 128Darkfield Microscopy 129Exercise: Darkfield Microscopy 1338. Properties of Polarized Light 135Overview 135The Generation of Polarized Light 135Demonstration: Producing Polarized Light with a Polaroid Filter 137Polarization by Reflection and Scattering 139Vectorial Analysis of Polarized Light Using a Dichroic Filter 139Double Refraction in Crystals 142Demonstration: Double Refraction by a Calcite Crystal 144Kinds of Birefringence 145Propagation of O and E Wavefronts in a Birefringent Crystal 146Birefringence in Biological Specimens 148Generation of Elliptically Polarized Light by Birefringent Specimens 1499. Polarization Microscopy 153Overview 153Optics of the Polarizing Microscope 155Adjusting the Polarizing Microscope 156Appearance of Birefringent Objects in Polarized Light 157Principles of Action of Retardation Plates and Three Popular Compensators 158Demonstration: Making a λ-Plate from a Piece of Cellophane 162Exercise: Determination of Molecular Organization in Biological Structures Using a Full Wave Plate Compensator 16710. Differential Interference Contrast Microscopy and Modulation Contrast Microscopy 173Overview 173The DIC Optical System 173Demonstration: The Action of a Wollaston Prism in Polarized Light 179Modulation Contrast Microscopy 190Exercise: DIC Microscopy 19411. Fluorescence Microscopy 199Overview 199Applications of Fluorescence Microscopy 201Physical Basis of Fluorescence 202Properties of Fluorescent Dyes 205Demonstration: Fluorescence of Chlorophyll and Fluorescein 206Autofluorescence of Endogenous Molecules 211Demonstration: Fluorescence of Biological Materials under UV Light 213Fluorescent Dyes and Proteins in Fluorescence Microscopy 213Arrangement of Filters and the Epi-Illuminator in the Fluorescence Microscope 218Objectives and Spatial Resolution in Fluorescence Microscopy 224Causes of High Fluorescence Background 225The Problem of Bleedthrough with Multiply Stained Specimens 227Quenching, Blinking, and Photobleaching 228Examining Fluorescent Molecules in Living Cells 23012. Fluorescence Imaging of Dynamic Molecular Processes 233Overview 233Modes of Dynamic Fluorescence Imaging 234Förster Resonance Energy Transfer 236Applications 244Fluorescence Recovery after Photobleaching 245TIRF Microscopy: Excitation by an Evanescent Wave 252Advanced and Emerging Dynamic Fluoresence Techniques 26113. Confocal Laser Scanning Microscopy 265Overview 265The Optical Principle of Confocal Imaging 267Demonstration: Isolation of Focal Plane Signals with a Confocal Pinhole 271Advantages of CLSM over Widefield Fluorescence Systems 273Criteria Defining Image Quality and the Performance of an Electronic Imaging System 275Confocal Adjustments and Their Effects on Imaging 277Photobleaching 286General Procedure for Acquiring a Confocal Image 286Performance Check of a Confocal System 288Fast (Real-Time) Imaging in Confocal Microscopy 288Spectral Analysis: A Valuable Enhancement for Confocal Imaging 295Optical Sectioning by Structured Illumination 297Deconvolution Microscopy 298Exercise: Effect of Confocal Variables on Image Quality 30414. Two-photon Excitation Fluorescence Microscopy 307Overview 307The Problem of Photon Scattering in Deep Tissue Imaging 308Two-Photon Excitation Is a Nonlinear Process 309Localization of Excitation 314Why Two-Photon Imaging Works 317Resolution 318Equipment 319Three-Photon Excitation 325Second Harmonic Generation Microscopy 32615. Superresolution Imaging 331Overview 331The RESOLFT Concept 333Single-Molecule Localization Microscopy 334Structured Illumination Microscopy 343Stimulated Emission Depletion (STED) Microscopy: Superresolution by PSF Engineering 34916. Imaging Living Cells with the Microscope 357Overview 357Labeling Strategies for Live-Cell Imaging 358Control of Illumination 361Control of Environmental Conditions 365Optics, Detectors, and Hardware 372Evaluating Live-Cell Imaging Results 384Exercise: Fluorescence Microscopy of Living Tissue Culture Cells 38417. Fundamentals of Digital Imaging 389Overview 389The Charge-Coupled Device (CCD Imager) 390CCD Designs 396Note: Interline CCD Imagers: The Design of Choice for Biomedical Imaging 398Back-Thinned Sensors 398EMCCD Cameras: High Performance Design for Greatest Sensitivity 399Scientific CMOS: The Next Generation of Scientific Imagers 400Camera Variables Affecting CCD Readout and Image Quality 401Six Terms Define Imaging Performance 404Aliasing 409Color Cameras 410Exercise: Evaluating the Performance of a CCD Camera 41118. Digital Image Processing 415Overview 415Preliminaries: Image Display and Data Types 416Histogram Adjustment 417Adjusting Gamma (γ) to Create Exponential LUTs 421Flat-Field Correction 421Image Processing With Filters 425Signal-to-Noise Ratio 432The Use of Color 438Images as Research Data and Requirements for Scientific Publication 442Exercise: Flat-Field Correction and Determination of S/N Ratio 448Appendix A: Answer Key to Exercises 451Appendix B: Materials for Demonstrations and Exercises 455Appendix C: Sources of Materials for Demonstrations and Exercises 463Glossary 465Microscopy Web Resources 509Recommended Reading 521References 523Index 531

“This should be provided to all beginning graduate students entering microscopy labs. It describes the complicated hardware of the system, while also explaining the physics principles of microscopy on a simplistic level for basic biologists. The authors achieve a perfect balance of theory and methods.”  (Doody’s, 15 November 2013)“It should be particularly useful to researchers getting started in the field of microscopy as well as seasoned professionals. Summing Up: Highly recommended. Graduate students, researchers/faculty, and professionals/practitioners.”  (Choice, 1 October 2013)“In summary, Fundamentals of Light Microscopy, Second Edition is a recommended starting point for the novice in microscopy and electronic imaging.”  (Journal of Biomedical Optics, 1 February 2013)