Optical Imaging Techniques in Cell Biology: 2nd Edition (Paperback) book cover

Optical Imaging Techniques in Cell Biology

2nd Edition

By Guy Cox

CRC Press

316 pages | 248 B/W Illus.

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Optical Imaging Techniques in Cell Biology, Second Edition covers the field of biological microscopy, from the optics of the microscope to the latest advances in imaging below the traditional resolution limit. It includes the techniques—such as labeling by immunofluorescence and fluorescent proteins—which have revolutionized cell biology. Quantitative techniques such as lifetime imaging, ratiometric measurement, and photoconversion are all covered in detail.

Expanded with a new chapter and 40 new figures, the second edition has been updated to cover the latest developments in optical imaging techniques. Explanations throughout are accurate, detailed, but as far as possible non-mathematical. This edition includes appendices with useful practical protocols, references, and suggestions for further reading. Color figures are integrated throughout.


Praise for the First Edition

“…represents an excellent resource for those wishing to gain a grounding in a broad range of optical techniques…written in a highly knowledgeable, enthusiastic and accessible manner…comprehensively covers virtually the entire field of microscopy. …a valuable addition to the bookshelf of many research laboratories…can quickly and easily provide a clear understanding of commonly used techniques and underlying concepts. Students, technicians, and researchers will find it useful whether they are intending to use the techniques, have been using the techniques for some time, or are merely curious to know more about what the techniques can offer the cell biologist.”

—Mark Prescott, Department of Biochemistry and Molecular Biology, Monash University, in Australian Biochemist, vol 38 no 3

Table of Contents

The Light Microscope

Lenses and Microscopes

The Back Focal Plane of a Lens

Good Resolution

Resolution: Rayleigh’s Approach


Add a Drop of Oil

Köhler Illumination

Optical Contrasting Techniques


Phase Contrast


Differential Interference Contrast

Hoffman Modulation Contrast

Which Technique Is Best?

Fluorescence and Fluorescence Microscopy

What Is Fluorescence?

What Makes a Molecule Fluorescent?

The Fluorescence Microscope

Optical Arrangement

Light Source

Filter Sets: Excitation Filter, Dichroic Mirror, and Barrier


Image Capture

Optical Layout for Image Capture

Color Recording

Additive Color Model

Subtractive Color Model

CCD Cameras

Frame-Transfer Array

Interline-Transfer Array

Back Illumination


Recording Color

Filter Wheels

Filter Mosaics

Three CCD Elements with Dichroic Beamsplitters

Boosting the Signal

The Confocal Microscope

The Scanning Optical Microscope

The Confocal Principle

Resolution and Point Spread Function

Lateral Resolution in the Confocal Microscope

Practical Confocal Microscopes

The Light Source: Lasers

Gas Lasers



Semiconductor Lasers

Supercontinuum Lasers

Laser Delivery

The Primary Beamsplitter

Beam Scanning

Pinhole and Signal Channel Configurations


The Digital Image

Pixels and Voxels


Spatial Sampling: The Nyquist Criterion

Temporal Sampling: Signal-to-Noise Ratio

Multichannel Images

Aberrations and Their Consequences

Geometrical Aberrations

Spherical Aberration



Field Curvature

Chromatic Aberration

Chromatic Difference of Magnification

Practical Consequences

Apparent Depth

Nonlinear Microscopy

Multiphoton Microscopy

Principles of Two-Photon Fluorescence

Theory and Practice

Lasers for Nonlinear Microscopy

Advantages of Two-Photon Excitation

Construction of a Multiphoton Microscope

Fluorochromes for Multiphoton Microscopy

Second Harmonic Microscopy


High-Speed Confocal Microscopy

Tandem Scanning (Spinning Disk) Microscopes

Petràn System

One-Sided Tandem Scanning Microscopes (OTSMS)

Microlens Array: The Yokogawa System

Slit-Scanning Microscopes

Multipoint-Array Scanners

Structured Illumination

Deconvolution and Image Processing


Deconvolving Confocal Images

Image Processing

Grayscale Operations

Image Arithmetic

Convolution: Smoothing And Sharpening

Three-Dimensional Imaging: Stereoscopy and Reconstruction

Surfaces: Two-And-A-Half Dimensions

Perception of the 3D World

Motion Parallax

Convergence and Focus of Our Eyes


Concealment of One Object by Another

Our Knowledge of the Size and Shape of Everyday Things

Light and Shade

Limitations of Confocal Microscopy


Three-Dimensional Reconstruction

Techniques That Require Identification of "Objects"

Techniques That Create Views Directly from Intensity Data

Simple Projections

Weighted Projection (Alpha Blending)

Green Fluorescent Protein

Structure and Properties of GFP

GFP Variants

Applications of GFP

Heat Shock

Cationic Lipid Reagents

DEAE–Dextran And Polybrene

Calcium Phosphate Coprecipitation



Gene Gun

Plants: Agrobacterium

Fluorescent Staining, Teresa Dibbayawan, Eleanor Kable, and Guy Cox


Types of Antibody

Raising Antibodies


Fluorescent Stains for Cell Components and Compartments

Quantitative Fluorescence

Fluorescence Intensity Measurements

Linearity Calibration



Ratio Imaging

Cell Loading

Membrane Potential

Fast-Response Dyes

Slow-Response Dyes

Fluorescence Recovery after Photobleaching

Advanced Fluorescence Techniques: FLIM, FRET, and FCS

Fluorescence Lifetime

Practical Lifetime Microscopy

Frequency Domain

Time Domain

Fluorescence Resonant Energy Transfer (FRET)

Why Use FRET?

Identifying And Quantifying Fret

Increase in Brightness of Acceptor Emission

Quenching of Emission from the Donor

Lifetime of Donor Emission

Protection from Bleaching of Donor

Fluorescence Correlation Spectroscopy (FCS)

Raster Image Correlation Spectroscopy

Evanescent Wave Microscopy

The Near-Field and Evanescent Waves

Total Internal Reflection Microscopy

Near-Field Microscopy

Beyond the Diffraction Limit

4Pi and Multiple-Objective Microscopy

Stimulated Emission Depletion (STED)

Structured Illumination

Stochastic Techniques

Super-Resolution Summary

Appendix A: Microscope Care and Maintenance


The Fluorescent Illuminator

Appendix B: Keeping Cells Alive under the Microscope, Eleanor Kable and Guy Cox





Appendix C: Antibody Labeling of Plant and Animal Cells: Tips and Sample Schedules, Eleanor Kable and Teresa Dibbayawan

Antibodies: Tips on Handling and Storage

Pipettes: Tips on Handling

Antibodies and Antibody Titrations


Immunofluorescence Protocol


Multiple Labeling and Different Samples

Plant Material


Diagram Showing Position of Antibodies on Multiwell Slide

Appendix D: Image Processing with ImageJ, Nuno Moreno


Different Windows in ImageJ

Image Levels

Colors and Look-Up


Size Calibration

Image Math


Stacks and 3D Representation

FFT and Image Processing

Macro Language in ImageJ


About the Author

Guy Cox is a professor within the Electron Microscopy Unit at the University of Sydney, Australia.

Subject Categories

BISAC Subject Codes/Headings:
MEDICAL / Biotechnology
SCIENCE / Life Sciences / Cytology