Optical Imaging Techniques in Cell Biology

About this book

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. Optical Imaging Techniques in Cell Biology, Second Edition includes appendices with useful practical protocols, references, and suggestions for further reading. Color figures are integrated throughout.

Contents

The Light Microscope
Lenses and Microscopes
The Back Focal Plane of a Lens
Good Resolution
Resolution: Rayleigh’s Approach
Abbe
Add a Drop of Oil
Köhler Illumination

Optical Contrasting Techniques
Darkfield
Phase Contrast
Polarization
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
Filter

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
Binning
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
Solid-State
Lasers
Semiconductor Lasers
Supercontinuum Lasers
Laser Delivery
The Primary Beamsplitter
Beam Scanning
Pinhole and Signal Channel Configurations
Detectors

The Digital Image
Pixels and Voxels
Contrast
Spatial Sampling: The Nyquist Criterion
Temporal Sampling: Signal-to-Noise Ratio
Multichannel Images

Aberrations and Their Consequences
Geometrical Aberrations
Spherical Aberration
Coma
Astigmatism
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
Summary

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
Deconvolution
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
Perspective
Concealment of One Object by Another
Our Knowledge of the Size and Shape of Everyday Things
Light and Shade
Limitations of Confocal Microscopy
Stereoscopy
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
Electroporation
Microinjection
Gene Gun
Plants: Agrobacterium

Fluorescent Staining, Teresa Dibbayawan, Eleanor Kable, and Guy Cox
Immunolabeling
Types of Antibody
Raising Antibodies
Labeling
Fluorescent Stains for Cell Components and Compartments

Quantitative Fluorescence
Fluorescence Intensity Measurements
Linearity Calibration
Measurement
Colocalization
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
Cleaning
The Fluorescent Illuminator

Appendix B: Keeping Cells Alive under the Microscope, Eleanor Kable and Guy Cox
Chambers
Light
Movement
Finally
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
Example
Immunofluorescence Protocol
Method
Multiple Labeling and Different Samples
Plant Material
Protocol
Diagram Showing Position of Antibodies on Multiwell Slide

Appendix D: Image Processing with ImageJ, Nuno Moreno
Introduction
Different Windows in ImageJ
Image Levels
Colors and Look-Up
Tables
Size Calibration
Image Math
Quantification
Stacks and 3D Representation
FFT and Image Processing
Macro Language in ImageJ
Index

Customer Reviews

Biography

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