Fundamentals of Light Microscopy and Electronic Imaging / Edition 2

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Fundamentals of Light Microscopy and Electronic Imaging, Second Edition provides a coherent introduction to the principles and applications of the integrated optical microscope system, covering both theoretical and practical considerations. It expands and updates discussions of multi-spectral imaging, intensified digital cameras, signal colocalization, and uses of objectives, and offers guidance in the selection of microscopes and electronic cameras, as well as appropriate auxiliary optical systems and fluorescent tags.

The book is divided into three sections covering optical principles in diffraction and image formation, basic modes of light microscopy, and components of modern electronic imaging systems and image processing operations. Each chapter introduces relevant theory, followed by descriptions of instrument alignment and image interpretation. This revision includes new chapters on live cell imaging, measurement of protein dynamics, deconvolution microscopy, and interference microscopy.

PowerPoint slides of the figures as well as other supplementary materials for instructors are available at a companion website:

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Editorial Reviews

From the Publisher
“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)

Doody's Review Service
Reviewer: Latha Malaiyandi, PhD (Midwestern University)
Description: This is a comprehensive overview of the new technology in light microscopy and electronic imaging introduced in the decade since the first edition was published in 2001.
Purpose: The goal is to provide cell biologists with a foundational basis in the tools and techniques required to image specimens of organisms and their components. The book starts with an introduction to fundamental principles of microscopy and each chapter adds in complexity to provide readers with more specific applications that may be relevant to their specific research goals.
Audience: The intended audience ranges from amateur microscopists to seasoned researchers who are looking to add sophisticated applications to their microscopy experiments.
Features: The book details the theoretical and practical knowledge of microscopy. It starts with basic light microscopy, but then delves into epi-fluorescence, confocal, and two-photon microscopy. It includes detailed photographs of internal microscopy hardware, beautiful examples of microscopy images of various specimens ranging from bacteria to mammalian tissues to whole organisms, such as Drosophila. Each chapter includes a list of recommended reviews on the topic. The diagrams and tables are well defined and provide an extensive comparison among different techniques, reagents, and instruments. The authors provide useful guidelines for selecting microscopes and microscope components based on user requirements without endorsing any one brand. In addition, they provide demonstrations of the care and maintenance of the instrument as well as useful applications (e.g. step-by-step instructions for calibrating a stage micrometer or aligning a mercury arc lamp). This new edition features exercises with answer keys, the materials and reagents needed for demonstrations, and an extensive list of web resources on microscopy.
Assessment: 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. Specifically, they discuss the theory behind microscopy, introduce practical applications, provide a historical basis for its development, and include their expert advice on the testing and purchasing of a microscopy system based on specific user requirements.

5 Stars! from Doody
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Product Details

  • ISBN-13: 9780471692140
  • Publisher: Wiley
  • Publication date: 10/16/2012
  • Edition description: New Edition
  • Edition number: 2
  • Pages: 552
  • Sales rank: 1,426,406
  • Product dimensions: 7.10 (w) x 10.10 (h) x 1.40 (d)

Meet 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.

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Table of Contents

Preface xi

Acknowledgments xii


Overview 1

Optical Components of the Light Microscope 1

Aperture and Image Planes in a Focused, Adjusted Microscope5

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


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


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 anEpi-Illuminator 43

Filters for Adjusting the Intensity and Wavelength ofIllumination 45

Effects of Light on Living Cells 50


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 Microscope76


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 Image85

Demonstration: Viewing the Airy Disk with a Pinhole Aperture85

Effect of Aperture Angle on Diffraction Spot Size 87

Diffraction by a Grating and Calculation of Its Line Spacing, D89

Demonstration: The Diffraction Grating 93

Abbé’s Theory for Image Formation in the Microscope94

A Diffraction Pattern Is Formed in the Rear Aperture of theObjective 97

Demonstration: Observing the Diffraction Image in the Rear FocalPlane of a Lens 98

Preservation of Coherence: Essential Requirement for ImageFormation 99

Exercise: Diffraction by Microscope Specimens 101


Overview 103

Numerical Aperture 103

Spatial Resolution 105

Depth of Field and Depth of Focus 109

Optimizing the Microscope Image: A Compromise between SpatialResolution and Contrast 109

Exercise: Resolution of Striae in Diatoms 112


Overview 115

Phase Contrast Microscopy 115

The Behavior of Waves from Phase Objects in Brightfi eldMicroscopy 119

Exercise: Determination of the Intracellular Concentration ofHemoglobin in Erythrocytes by Phase Immersion Refractometry 128

Darkfi eld Microscopy 129

Exercise: Darkfi eld Microscopy 133


Overview 135

The Generation of Polarized Light 135

Demonstration: Producing Polarized Light with a Polaroid Filter137

Polarization by Refl ection and Scattering 139

Vectorial Analysis of Polarized Light Using a Dichroic Filter139

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 Crystal146

Birefringence in Biological Specimens 148

Generation of Elliptically Polarized Light by BirefringentSpecimens 149


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 PopularCompensators 158

Demonstration: Making a λ-Plate from a Piece of Cellophane162

Exercise: Determination of Molecular Organization in BiologicalStructures Using a Full Wave Plate Compensator 167


Overview 173

The DIC Optical System 173

Demonstration: The Action of a Wollaston Prism in PolarizedLight 179

Modulation Contrast Microscopy 190

Exercise: DIC Microscopy 194


Overview 199

Applications of Fluorescence Microscopy 201

Physical Basis of Fluorescence 202

Properties of Fluorescent Dyes 205

Demonstration: Fluorescence of Chlorophyll and Fluorescein206

Autofl uorescence of Endogenous Molecules 211

Demonstration: Fluorescence of Biological Materials under UVLight 213

Fluorescent Dyes and Proteins in Fluorescence Microscopy 213

Arrangement of Filters and the Epi-Illuminator in theFluorescence Microscope 218

Objectives and Spatial Resolution in Fluorescence Microscopy224

Causes of High Fluorescence Background 225

The Problem of Bleedthrough with Multiply Stained Specimens227

Quenching, Blinking, and Photobleaching 228

Examining Fluorescent Molecules in Living Cells 230


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


Overview 265

The Optical Principle of Confocal Imaging 267

Demonstration: Isolation of Focal Plane Signals with a ConfocalPinhole 271

Advantages of CLSM over Widefield Fluorescence Systems 273

Criteria Defining Image Quality and the Performance of anElectronic 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 Imaging295

Optical Sectioning by Structured Illumination 297

Deconvolution Microscopy 298

Exercise: Effect of Confocal Variables on Image Quality 304


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


Overview 331

The RESOLFT Concept 333

Single-Molecule Localization Microscopy 334

Structured Illumination Microscopy 343

Stimulated Emission Depletion (STED) Microscopy: Superresolutionby PSF Engineering 349


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 Cells384


Overview 389

The Charge-Coupled Device (CCD Imager) 390

CCD Designs 396

Note: Interline CCD Imagers: The Design of Choice for BiomedicalImaging 398

Back-Thinned Sensors 398

EMCCD Cameras: High Performance Design for Greatest Sensitivity399

Scientific CMOS: The Next Generation of Scientific Imagers400

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


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 ScientificPublication 442

Exercise: Flat-Field Correction and Determination of S/N Ratio448

Appendix A: Answer Key to Exercises 451

Appendix B: Materials for Demonstrations and Exercises 455

Appendix C: Sources of Materials for Demonstrations andExercises 463

Glossary 465

Microscopy Web Resources 509

Recommended Reading 521

References 523

Index 531

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