Cox G. Optical Imaging Techniques in Cell Biology

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edited by Guy Cox

with contributions by Eleanor Kable, Nuno Moreno and Teresa Dibbayawan

Published November 2006 by CRC Press / Taylor & Francis. 344pp; ISBN: 0‑8493‑3919‑7

Using a highly accessible style and format, this book:

  • Provides an understanding of the underlying principles, benefits, and limitations of optical techniques currently used in cell biology
  • Presents a cogent, sophisticated discussion without relying on complex mathematics
  • Addresses basic concepts in imaging such as contrasting techniques, fluorescence, and the correction of aberrations
  • Includes multiple appendices on practical maintenance, cell handling, labeling, and image manipulation


Contents

Table of Contents

  • Introduction – the optical microscope in cell biology
  • Brief historical overview from Robert Hooke (cells, 1685) through Swammerdam, van Leeuwenhoek (sperm, bacteria, blood cells), Robert Browne (nucleus), Schwann & Schleiden (cell theory), Lister (corrected objectives), Abbe, etc.

Chapter 1 - The light microscope

  • Lenses and microscopes
  • Good resolution
    • Resolution - Rayleigh’s approach
    • Abbe
    • Add a drop of oil ...
  • Köhler Illumination

Chapter 2 – Optical contrasting techniques

Chapter 3 - Fluorescence and fluorescence microscopes

  • What is fluorescence?
    • How molecules fluoresce
    • What makes a molecule fluorescent?
    • Pathways for de-excitation – photobleaching
    • Quantum yield and extinction coefficient
  • The fluorescence microscope

Chapter 4 – Image capture

Chapter 5 - The confocal microscope

  • The confocal principle
  • Practical confocal microscopes
    • The light source – lasers
    • Gas lasers
    • Solid state lasers
    • Semiconductor lasers
    • Laser delivery
    • The primary beamsplitter
    • Beam Scanning
    • Pinhole and signal channel configurations
    • Detectors

Chapter 6 – The digital image

  • Pixels and voxels
  • Contrast
  • Spatial sampling - the Nyquist criterion
  • Temporal sampling - signal to noise ratio
  • Multichannel images

Chapter 7 – Aberrations and their consequences

Chapter 8 - Non linear microscopy

  • Multiphoton microscopy
    • Principles of multiphoton fluorescence
    • Theory and practice
    • Lasers for non-linear microscopy
    • Advantages of two-photon excitation
    • Construction of a multiphoton microscope
    • Fluorochromes for multiphoton microscopy
  • Harmonic microscopy

Chapter 9 - High-speed confocal

  • Spinning disk
    • Tandem scanning microscope
    • One-sided TSM
    • Microlens spinning disk systems
    • Costs and benefits of disk-based systems
  • Slit scanners
    • True slit scanning systems
    • Spot scanning, slit detection
  • Multifocus
  • Structured illumination

Chapter 10 - Deconvolution and image processing Guy Cox & Nuno Moreno

  • 3D deconvolution of wide-field images
    • Overview – the problem
    • Inverse filters
    • Iterative approaches
  • Improving the confocal image
  • Convolution filters for smoothing and sharpening

Chapter 11 - Three dimensional imaging - stereoscopy & reconstruction

  • Surfaces – 2½ dimensions
  • Perception of the 3D world
    • Limitations of confocal microscopy
  • Stereoscopy
  • Three-dimensional reconstruction
    • Techniques which require objects to be identified
  • Techniques which create views directly from intensity data
    • Simple projections
    • Weighted projection (alpha blending)

Chapter 12 - GFP, its relatives and derivatives

  • The discovery of GFP
  • GFP – structure and function
  • GFP variants
    • Man-made mutants
    • Wild variants
    • Techniques for transfection

Chapter 13 – Fluorescent staining Guy Cox, Teresa Dibbayawan & Eleanor Kable

  • Immunolabelling
    • Basics of the immune system
    • Types of antibodies
    • Raising antibodies
    • Labelling
  • Other fluorescent probes, stains and labels
  • Vital dyes
    • Cell-permeant dyes for organelles and compartments
    • Membrane tracer dyes

Chapter 14 - Quantitative fluorescence

Chapter 15 – Advanced fluorescence techniques

Chapter 16 - Breaking the diffraction limit

Appendices

  • Appendix 1 – Guy Cox. Microscope care and maintenance
  • Appendix 2 – Guy Cox & Eleanor Kable. Keeping cells alive under the microscope.
  • Appendix 3 – Teresa Dibbayawan & Eleanor Kable. Sample protocols for immuno­labelling of plant and animal cells
  • Appendix 4 – Nuno Moreno. Image processing with Image J
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