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Bioimaging: Current Concepts in Light & Electron Microscopy

Author(s): Douglas E Chandler, PhD, Arizona State University
Robert W. Roberson, PhD, Arizona State University
Details:
  • ISBN-13: 9780763738747
  • ISBN-10:0763738743
  • Hardcover    440 pages      © 2009
Price: International Sales $225.95 US List
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The development of microscopy revolutionized the world of cell and molecular biology as we once knew it and will continue to play an important role in future discoveries. Bioimaging: Current Concepts in Light and Electron Microscopy is the optimal text for any undergraduate or graduate bioimaging course, and will serve as an important reference tool for the research scientist. This unique text covers, in great depth, both light and electron microscopy, as well as other structure and imaging techniques like x-ray crystallography and atomic force microscopy.  Written in a user-friendly style and covering a broad range of topics, Bioimaging describes the state-of-the-art technologies that have powered the field to the forefront of cellular and molecular biological research.    

Features & Benefits

Provides the conceptual framework for a wide array of imaging techniques in a concise and understandable format using examples that have contributed important information to the study of cellular structure and function.

Addresses and compares a wide variety of both classical and state of the art techniques in bioimaging.  Also includes two chapters on the history of microscopy and image reproduction.

Describes the state-of-the-art technologies that have powered bioimaging to the forefront of cellular and molecular biology research.  Includes  such techniques as rapid freezing, cryoelectron microscopy, electronic imaging, single molecule fluorescence microscopy, atomic force microscopy, electron microscopy tomography, calcium ratio imaging microscopy and immunocytochemistry. 

Applicable Courses

Appropriate for Bioimaging courses of any kind at any level (both graduate and undergraduate) including courses that cover techniques used in molecular and cellular biology.

1. History of Microscopy
    1.1  Development of the Light Microscope

    1.2  Development of the Electron Microscope

    1.3  Development of Other Imaging Technologies

    1.4  Development of Specimen Preparation Methods for Light and Electron Microscopy

    1.5  Conclusion

    1.6  References and Suggested Reading

 

2. History of Microscopic Image Reproduction

    2.1  Photographic Darkroom Procedures

    2.2  Digital Photography

    2.3  Low Volume Printing of Electronic Image Files

    2.4  High Speed Commercial Printing

    2.4  Color Reproduction

    2.5  References and Suggested Reading

 

3. Preparation of Specimens for Light and Electron Microscopy

    3.1  Fixation

    3.2  En Bloc Staining

    3.3  Dehydration

    3.4  Infiltration and Embedding

    3.5  Microtomy—Cutting Sections

    3.6  Staining

    3.7  Methods That Facilitate the Overall Tissue Processing

    3.8  Non-Standard Methods for Tissue Processing

    3.9  Artifacts of Tissue Processing

    3.10 Interpretation of Three-Dimensional Tissue Structure Using Two-Dimensional Sections

    3.11 Storage and Documentation of Microscopic Specimens

    3.12 References and Suggested Reading

 

4. A Brief Introduction to Cell Structure

    4.1  The Animal Cell

    4.2  The Cell Surface

    4.3  Cell Organelles

    4.4  Plant Cells

    4.5  Fungal Cells

    4.6  Prokaryotic Cells

    4.7  References and Suggested Readings

 

5. Electromagnetic Radiation and Its Interaction With Matter

    5.1  Interaction of Electromagnetic Radiation With Specimens

    5.2  Speed of light

    5.3  Reflection

    5.4  Refraction

    5.5  Diffraction

    5.6  Interference

    5.7  Polarization

    5.8  Absorption and Emission of Radiation

    5.9  Qualities of Electromagnetic Radiation Summarized

    5.10 References and Suggested Readings

 

6. The Light Microscope and Image Formation

    6.1  Image Formation Requires Diffraction and Interference

    6.2  Lens Aberrations

    6.3  Objective Lenses Oculars

    6.4  Oculars

    6.5  Light Sources and Lamp Houses

    6.6  The Specimen Stage

    6.7  Design Plan of the Compound Light Microscope

    6.8  Light Paths in the Compound Microscope

    6.9  Kohler Illumination

    6.10 Infinity-Corrected Optics

    6.11 Prisms and Beamsplitters

    6.12 References and Suggested Readings

 

7. Phase, Interference and Polarization Methods for Optical Contrast

    7.1  Vital Dyes

    7.2  Phase Contrast Microscopy

    7.3  Darkfield Microscopy

    7.4  Polarization Microscopy

    7.5  Differential Interference Contrast Microscopy

    7.6  Hoffman Interference Contrast

    7.7  References and Suggested Reading

 

8. The Transmission Electron Microscope:

    8.1  Illumination System

    8.2  Specimen Chamber and Control System

    8.3  Imaging System

    8.4  Vacuum System

    8.5  Practical Guide for Getting Started

    8.6  References and Suggested Reading

 

9. The Scanning Electron Microscope

    9.1  The Systems and Principles of the SEM

    9.2  Sample Preparation

    9.3  Analytical Microscopy

    9.4  Additional Modes of SEM Analysis and Operations

    9.5  Practical Guide for Getting Started

    9.6  References and Suggested Reading

 

10. Cryogenic Techniques in Electron Microscopy

   10.1  Freezing Methods Using Cryoprotectants

   10.2  Ultrarapid Freezing by Immersion

   10.3  Propane Jet Freezing

   10.4  Metal Mirror Freezing

   10.5  High Pressure Freezing

   10.6  Cryofixation Avoids Artifacts of Chemical Fixation But Can Cause Freezing Damage

   10.7  Freeze Substitution

   10.8  Freeze Fracture and Replica Production

   10.9  Imaging and Presenting Freeze Fracture Replicas

   10.10 Live Cells Can Be Rapidly Frozen and Freeze Fractured

   10.11 Quick-Freezing, Deep-Etching and Rotary Shadowing (QF-DE-RS)

   10.12 References and Suggested Reading

 

11. Video Microscopy and Electronic Imaging

   11.1  Video Cameras—The Old Tube Type

   11.2  Solid State Cameras

   11.3  Solid State Color Cameras

   11.4  Digital Image Processing

   11.5  Signal to Noise Ratio

   11.6  Storage of Electronic Signals

   11.7  Video Cassette Recorders

   11.8  Playback of Video and Electronic Images

   11.9  Digital Video Processing

   11.10 References and Suggested Reading

 

12. Fluorescence Microscopy

   12.1  The Molecular Basis of Fluorescence

   12.2  Optical Filters

   12.3  Fluorescent Dyes

   12.4  Quantum Dots

   12.5  Using Multiple Dyes

   12.6  Autofluorescence and Background Fluorescence

   12.7  Laser Scanning Confocal Microscopy (LSCM)

   12.8  Lasers

   12.9  Delivery of Laser Light to the Scanning Head

   12.10 Scanning the Beam Onto the Specimen

   12.11 Light Collection and Data Analysis

   12.12 Line Scanning and Spinning Disk Confocal Microscopy

   12.13 Multi-Photon Confocal Microscopy

   12.14 Formats for Presentation of Three Dimensional Data Sets

   12.15 References and Suggested Reading

 

13. Microscopic Localization and Dynamics of Biological Molecules

   13.1  Cytochemistry

   13.2  Autoradiography

   13.3  Use of Fluorescent Probes

   13.4  Immunocytochemistry

   13.5  Fluorescent Proteins (Green and Otherwise)

   13.6  In Situ Hybridization and FISH

   13.7  Multi-spectral Imaging and Microscopy and Chromosome Painting

   13.8  Localization of Lipids, Carbohydrates and Other Molecules based on Chemical Affinity

   13.9  Single Molecule Fluorescence

   13.10 Detection of Molecular Motions and Interactions: FRET, FRAP, FCS, Fluorescence Polarization and Speckle Microscopy

   13.11 Single Particle Tracking Using Video Microscopy

   13.12 Optical Tweezers—the Laser Trapping of Small Organelles, Particles and Motile Cells

   13.13 Fluorescence Activated Cell Sorting (FACS)

   13.14 References and Suggested Reading

 

14. Imaging Ions and Intracellular Messengers

   14.1  Precipitation Techniques

   14.2  Rapid Freezing Techniques

   14.3  Calcium Detection in Live Cells Using Fluorescent and Luminescent Probes

   14.4  Calcium Homeostasis

   14.5  Fluorescent Calcium Chelators—a New Generation of Probes

   14.6  Fura-2 and Indo-1: Second Generation Calcium Probes

   14.7  Requirements for Live Cell Imaging

   14.8  Quantitation of Fluorescence Signals

   14.9  Calcium Signals Visualized by Fluorescent Probes

   14.10 Fluorescent Probes for Other Ions

   14.11 Potential-Sensitive Probes

   14.12 Cyclic Nucleotide and Protein Kinase Probes

   14.13 References and Suggested Reading

 

15. Imaging Macromolecules and Supermolecular Complexes

   15.1  Rotary Platinum Shadowing

   15.2  Negative Staining

   15.3  Transmission EM of Frozen Macromolecules

   15.4  Electron Microscope Tomography

   15.5  X-Ray and Electron Diffraction Methods

   15.6  Scanning Probe Microscopy Near Field Microscopy

   15.7  Near Field Microscopy

   15.8  References and Suggested Reading

 

16. Image Processing and Presentation

   16.1  Start With the Right Specimen and Microscopy Technique

   16.2  Getting the Most Out of Your Microscopy

   16.3  Post-Production Analysis and Optimization of Images

   16.4  When is Image Processing Reasonable?

   16.5  Obtaining Quantitative Data From Images

   16.6  Motion Analysis

   16.7  Three-Dimensional Reconstructions

   16.8  Methods Used for Presentation of Images

   16.9  References and Suggested Reading

 


Douglas E Chandler, PhD-Arizona State University

Dr. Chandler received his undergraduate education in chemistry at the University of Rochester, and his graduate education in biochemistry and physiology at the John Hopkins School of Medicine and the University of California at San Francisco.  After postdoctoral fellowships in San Francisco and at University College London School of Medicine, he assumed his current position at Arizona State University in 1980.  His research specializations include the roll of egg extra cellular matrices in fertilization, the structure and activity of sperm activation proteins and membrane structural changes during exocytosis and membrane fusion.  He has published over 70 research articles in the professional literature and has been the recipient of NSF, NIH, and private foundation research grants.  As Director of the W.M. Keck Bioimaging Laboratory he has taught bioimaging courses for over 10 years and has previously served on the editorial board of Microscopy Research and Technique.

Robert W. Roberson, PhD-Arizona State University