Light and Video Microscopy - 2nd Edition - ISBN: 9780124114845, 9780124115361

Light and Video Microscopy

2nd Edition

Authors: Randy Wayne
eBook ISBN: 9780124115361
Hardcover ISBN: 9780124114845
Imprint: Academic Press
Published Date: 2nd January 2014
Page Count: 366
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The purpose of this book is to provide the most comprehensive, easy-to-use, and informative guide on light microscopy. Light and Video Microscopy will prepare the reader for the accurate interpretation of an image and understanding of the living cell. With the presentation of geometrical optics, it will assist the reader in understanding image formation and light movement within the microscope. It also provides an explanation of the basic modes of light microscopy and the components of modern electronic imaging systems and guides the reader in determining the physicochemical information of living and developing cells, which influence interpretation.

Key Features

  • Brings together mathematics, physics, and biology to provide a broad and deep understanding of the light microscope
  • Clearly develops all ideas from historical and logical foundations
  • Laboratory exercises included to assist the reader with practical applications
  • Microscope discussions include: bright field microscope, dark field microscope, oblique illumination, phase-contrast microscope, photomicrography, fluorescence microscope, polarization microscope, interference microscope, differential interference microscope, and modulation contrast microscope


Cell biologists, neurobiologists, reproductive biologists, plant biologists, microbiologists, fungal biologists, veterinarians, forensic scientists, food scientists, horticulturalists, biochemists, physicists, engineers, and anyone using a light or video microscope

Table of Contents


Preface to the Second Edition

Preface to the First Edition

Chapter 1. The Relation Between the Object and the Image

Luminous and Nonluminous Objects

Object and Image

Theories of Vision

Light Travels in Straight Lines

Images Formed in a Camera Obscura: Geometric Considerations

Where Does Light Come From?

How Can the Amount of Light Be Measured?

Web Resources

Chapter 2. The Geometric Relationship Between Object and Image

Reflection by a Plane Mirror

Reflection by a Curved Mirror

Reflection from Various Sources

Images Formed by Refraction at a Plane Surface

Images Formed by Refraction at a Curved Surface

Fermat’s Principle

Optical Path Length

Lens Aberrations

Geometric Optics and Biology

Geometric Optics of the Human Eye

Web Resources

Chapter 3. The Dependence of Image Formation on the Nature of Light

Christiaan Huygens and the Invention of the Wave Theory of Light

Thomas Young and the Development of the Wave Theory of Light

James Clerk Maxwell and the Wave Theory of Light

Ernst Abbe and the Relationship of Diffraction to Image Formation

Resolving Power and the Limit of Resolution


Web Resources

Chapter 4. Bright-Field Microscopy

Components of the Microscope

The Optical Paths of the Light Microscope

Using the Bright-Field Microscope

Depth of Field

Out-of-Focus Contrast

Uses of Bright-Field Microscopy

Care and Cleaning of the Light Microscope

Web Resources

Chapter 5. Photomicrography

Setting up the Microscope for Photomicrography

Scientific History of Photography

General Nature of the Photographic Process

The Resolution of the Film

Exposure and Composition

The Similarities between Film and the Retina

Web Resources

Chapter 6. Methods of Generating Contrast

Dark-Field Microscopy

Rheinberg Illumination

Oblique Illumination

Phase-Contrast Microscopy

Oblique Illumination Reconsidered

Annular Illumination

Chapter 7. Polarization Microscopy

What is Polarized Light?

Use an Analyzer to Test for Polarized Light

Production of Polarized Light

Influencing Light

Design of a Polarizing Microscope

What is the Molecular Basis of Birefringence?

Interference of Polarized Light

The Origin of Colors in Birefringent Specimens

Use of Compensators to Determine the Magnitude and Sign of Birefringence

Crystalline versus Form Birefringence

Orthoscopic versus Conoscopic Observations

Reflected Light Polarization Microscopy

Uses of Polarization Microscopy

Optical Rotatory (or Rotary) Polarization and Optical Rotatory (or Rotary) Dispersion

Web Resources

Chapter 8. Interference Microscopy

Generation of Interference Colors

The Relationship of Interference Microscopy to Phase-Contrast Microscopy

Quantitative Interference Microscopy: Determination of the Refractive Index, Mass, Concentration of Dry Matter, Concentration of Water, and Density

Source of Errors When Using an Interference Microscope

Making a Coherent Reference Beam

Double-Beam versus Multiple-Beam Interference

Interference Microscopes Based on a Mach–Zehnder Type Interferometer

Interference Microscopes Based on Polarized Light

The Use of Transmission Interference Microscopy in Biology

Reflection–Interference Microscopy

Uses of Reflection–Interference Microscopy in Biology

Web Resources

Chapter 9. Differential Interference Contrast (DIC) Microscopy

Design of a Transmitted Light Differential Interference Contrast Microscope

Interpretation of a Transmitted Light Differential Interference Contrast Image

Design of a Reflected Light Differential Interference Contrast Microscope

Interpretation of a Reflected Light Differential Interference Contrast Image

Chapter 10. Amplitude Modulation Contrast Microscopy

Hoffman Modulation Contrast Microscopy

Reflected Light Hoffman Modulation Contrast Microscopy

The Single-Sideband Edge Enhancement Microscope

Web Resources

Chapter 11. Fluorescence Microscopy

Discovery of Fluorescence

Physics of Fluorescence

Design of a Fluorescence Microscope

Fluorescence Probes

Pitfalls and Cures in Fluorescence Microscopy

Web Resources

Chapter 12. Various Types of Microscopes and Accessories

Confocal Microscopes

Total Internal Reflectance Fluorescence Microscopy

Superresolution With Wide-Field Microscopes

Fluorescence Resonance Energy Transfer

Fluorescence Redistribution After Photobleaching

Laser Microbeam Microscope

Optical Tweezers

Laser Capture Microdissection

Laser Doppler Microscope

Centrifuge Microscope

X-Ray Microscope

Infrared Microscope

Nuclear Magnetic Resonance Imaging Microscope

Stereo Microscopes

Scanning Probe Microscopes

Acoustic Microscope

Horizontal and Traveling Microscopes

Microscopes for Children

Microscope Accessories

Web Resources

Chapter 13. Video and Digital Microscopy

The Value of Video and Digital Microscopy

Video and Digital Cameras: The Optical-to-Electrical Signal Converters

Monitors: Conversion of an Electronic Signal into an Optical Signal

Storage of Video and Digital Images

Connecting a Video System

Web Resources

Chapter 14. Image Processing and Analysis

Analog Image Processing

Digital Image Processing

Enhancement Functions of Digital Image Processors

Analysis Functions of Digital Image Processors

Toupview Digital Image Processing Software

The Ethics of Digital Image Processing

Web Resources

Chapter 15. Laboratory Exercises

Laboratory 1: The Nature of Light and Geometric Optics

Laboratory 2: Physical Optics

Laboratory 3: The Bright-Field Microscope and Image Formation

Laboratory 4: Phase-Contrast Microscopy, Dark-Field Microscopy, Rheinberg Illumination, and Oblique Illumination

Laboratory 5: Fluorescence Microscopy

Laboratory 6: Polarized Light

Laboratory 7: Polarizing Light Microscopy

Laboratory 8: Interference Microscopy

Laboratory 9: Differential Interference Contrast Microscopy and Hoffman Modulation Contrast Microscopy

Laboratory 10: Video and Digital Microscopy and Analog and Digital Image Processing

Web Resources


Appendix I. Light Microscopy: A Retrospective

Web Resource

Appendix II. A Final Exam

Appendix III. A Microscopist’s Model of the Photon


Color Plates


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About the Author

Randy Wayne

Randy O. Wayne is a plant cell biologist at Cornell University notable for his work on plant development. In particular, along with his colleague Peter K. Hepler, Wayne established the powerful role of calcium in regulating plant growth; accordingly, their 1985 article, Calcium and plant development, was cited by at least 405 subsequent articles to earn the "Citation Classic" award from Current Contents magazine and has been cited by hundreds more since 1993. He is an authority on how plant cells sense gravity through pressure, on the water permeability of plant membranes, light microscopy, as well as the effects of calcium on plant development. He has published over 50 articles and is the author of another book, Light and Video Microscopy.

Affiliations and Expertise

Department of Plant Biology Cornell University, Ithaca, NY, USA


"As such, this book would be an excellent undergraduate-level text on optical microscopy for biologists. It is also valuable to anyone using a light microscope." --Carol Bayles, Cornell University, Ithaca, NY, USA