2nd Edition

Optical Imaging Techniques in Cell Biology

By Guy Cox Copyright 2012
    316 Pages 248 B/W Illustrations
    by CRC Press

    316 Pages 248 B/W Illustrations
    by CRC Press

    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.

    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


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

    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