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.
TOC:
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