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Fundamentals of Light Microscopy and Electronic Imaging
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Table of Contents

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 Infi nity 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 Specifi cations 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 Abbe?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 Brightfi eld Microscopy 119 Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry 128 Darkfi eld Microscopy 129 Exercise: Darkfi eld 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 Refl ection 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 Autofl uorescence 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 Foerster 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

About the Author

DOUGLAS B. MURPHY supervises core facilities inmicroscopy and histology at the new HHMI Janelia Farm ResearchCampus in Ashburn, Virginia. An Adjunct Professor of Cell Biologyat Johns Hopkins School of Medicine in Baltimore, Maryland, Dr.Murphy helped establish the School of Medicine Microscope Facilitythere, which he supervised until 2006. MICHAEL W. DAVIDSON is an assistant scholar/scientistaffiliated with the National High Magnetic Field Laboratory and theDepartment of Biological Science at Florida State University wherehe is involved in developing educational websites. His digitalimages and photomicrographs have graced the covers of over 2,000publications.

Reviews

This should be provided to all beginning graduatestudents entering microscopy labs. It describes the complicatedhardware of the system, while also explaining the physicsprinciples of microscopy on a simplistic level for basicbiologists. The authors achieve a perfect balance of theory andmethods. (Doody s, 15 November 2013) It should be particularly useful to researchers gettingstarted in the field of microscopy as well as seasonedprofessionals. Summing Up: Highly recommended. Graduate students,researchers/faculty, and professionals/practitioners. (Choice, 1 October 2013) In summary, Fundamentals of Light Microscopy, SecondEdition is a recommended starting point for the novice inmicroscopy and electronic imaging. (Journal ofBiomedical Optics, 1 February 2013)

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