Keywords:
Video microscopy.
;
Electronic books.
Description / Table of Contents:
The previous edition of this book marked the shift in technology from video to digital camera use with microscope use in biological science. This new edition presents some of the optical fundamentals needed to provide a quality image to the digital camera. Specifically, it covers the fundamental geometric optics of finite- and infinity-corrected microscopes, develops the concepts of physical optics and Abbe's theory of image formation, presents the principles of Kohler illumination, and finally reviews the fundamentals of fluorescence and fluorescence microscopy. The second group of chapters deals with digital and video fundamentals: how digital and video cameras work, how to coordinate cameras with microscopes, how to deal with digital data, the fundamentals of image processing, and low light level cameras. The third group of chapters address some specialized areas of microscopy that allow sophisticated measurements of events in living cells that are below the optical limits of resolution. * Expands coverage to include discussion of confocal microscopy not found in the previous edition * Includes "traps and pitfalls" as well as laboratory exercises to help illustrate methods.
Type of Medium:
Online Resource
Pages:
1 online resource (626 pages)
Edition:
3rd ed.
ISBN:
9780080544342
Series Statement:
Issn Series ; v.Volume 114
URL:
https://ebookcentral.proquest.com/lib/geomar/detail.action?docID=296622
DDC:
502.8/2
Language:
English
Note:
Front Cover -- Methods in Cell Biology -- Copyright Page -- Contents -- Contributors -- Preface -- Chapter 1: Microscope Basics -- I. Introduction -- II. How Microscopes Work -- A. The Finite Tube-Length Microscope -- B. Infinity Optics Microscopes -- III. Objective Basics -- A. Types of Objectives -- B. "Mixing and Matching" Objectives -- C. Coverslip Selection -- IV. Mounting Video Cameras on the Microscope -- A. Basic Considerations -- B. Empty Magnification -- C. Camera Pixel Number and Resolution -- Reference -- Chapter 2: The Optics of Microscope Image Formation -- I. Introduction -- II. Physical Optics: The Superposition of Waves -- III. Huygens' Principle -- IV. Young's Experiment: Two-Slit Interference -- V. Diffraction from a Single Slit -- VI. The Airy Disk and the Issue of Microscope Resolution -- VII. Fourier or Reciprocal Space: The Concept of Spatial Frequencies -- VIII. Resolution of the Microscope -- IX. Resolution and Contrast -- X. Conclusions -- XI. Appendix I -- A. Fourier Series -- XII. Appendix II -- A. Kirchoff's Scalar Theory of Diffraction: Recasting Huygen's Principle in an Electrodynamic Context -- B. Generalizing the Problem -- C. Scalar Spherical Waves -- D. Green's Theorem -- E. Solution for a Plane -- F. Huygens' Principle -- XIII. Appendix III -- A. Diffraction by a Circular Aperture from Which the Airy Disk Comes -- Acknowledgments -- References -- Chapter 3: Proper Alignment of the Microscope -- I. Key Components of Every Light Microscope -- A. Light Source -- B. Lamp Collector -- C. Diffusers and Filters -- D. Field Diaphragm -- E. Condensers -- F. Aperture Diaphragm -- G. Condenser Carrier or Substage -- H. The Specimen Stage -- I. The Objective -- J. Revolving Nosepiece -- K. Infinity Space -- L. Tube, Eyepiece, and Video Adapters -- II. Koehler Illumination.
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A. Aligning the Microscope for Koehler Illumination -- B. What Are the Benefits of Koehler Illumination? -- C. Resolution and Contrast -- Chapter 4: Mating Cameras to Microscopes -- I. Introduction -- II. Optical Considerations -- Chapter 5: Fundamentals of Fluorescence and Fluorescence Microscopy -- I. Introduction -- II. Light Absorption and Beer's Law -- III. Atomic Fluorescence -- IV. Organic Molecular Fluorescence -- V. Excited State Lifetime and Fluorescence Quantum Efficiency -- VI. Excited State Saturation -- VII. Nonradiative Decay Mechanisms -- VIII. Fluorescence Resonance Energy -- IX. Fluorescence Depolarization -- X. Measuring Fluorescence in the Steady State -- XI. Construction of a Monochromator -- XII. Construction of a Photomultiplier Tube -- XIII. Measuring Fluorescence in the Time-Domain -- A. Boxcar-Gated Detection Method -- B. Streak Camera Method -- C. Photon Correlation Method -- D. Note on the Process of "Deconvolution -- E. Phase Modulation Method -- XIV. Filters for the Selection of Wavelength -- XV. The Fluorescence Microscope -- XVI. The Power of Fluorescence Microscopy -- Some Useful Web Sources -- Acknowledgments -- References -- Chapter 6: Fluorescent Protein Applications in Microscopy -- I. Introduction -- II. The Identification of Green Fluorescent Protein -- III. Formation of the GFP Chromophore -- IV. The Structure of GFP -- V. Mutagenesis to Alter the Properties of GFP -- VI. Imaging FPs -- A. Components of the Optical System -- B. Reducing Unwanted Flurescence -- C. Selecting Appropriate Filters and Chromatic Mirrors -- D. Selecting the Appropriate FP -- E. Constructing FP Fusions -- F. A.Note on Fixation of FPs -- G. Time-Lapse Imaging of FPs -- VII. Applications of FP Imaging -- A. Multiwavelength Imaging of FPs -- B. Monitoring Intracellular Environments Using FPs.
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C. Monitoring Protein-Protein Interactions In Vivo -- D. Protein Dynamic Measurements Using FP Photobleaching and Activation -- VIII. Conclusion -- References -- Chapter 7: Live-Cell Fluorescence Imaging -- I. Introduction -- II. Preparing a Specimen for Fluorescence Live-Cell Imaging -- A. Choice of Fluorophore -- B. Mounting the Specimen for Viewing -- C. Maintaining Temperature -- D. Cell Culture Media for Imaging -- E. Preventing Photobleaching -- III. Choice of Microscope -- A. The Microscope Stand -- B. Mode of Fluorescence Microscopy -- IV. Wide-Field Illumination of the Specimen -- A. Light Source -- B. Diaphragms -- C. Filter Sets -- D. Automation of Filter Selection -- V. Choosing the Best Objective Lens for Your Application -- A. NA: Resolution and Brightness -- B. Magnification -- C. Correction for Aberrations -- D. Phase and DIC Objective Lenses -- VI. Acquiring Digital Images Over Time -- A. Cameras -- B. Shutters -- C. Maintaining Focus -- VII. nD Imaging -- VIII. Verifying Cell Health and Troubleshooting Sick Cells -- IX. Conclusion -- Acknowledgments -- References -- Chapter 8: Working with Classic Video -- I. Introduction -- II. The Black and White Video Signal -- III. Adjusting the Camera and Video Monitor -- A. Camera Controls -- B. Monitor Control -- IV. Practical Aspects of Coordinately Adjusting Camera and Monitor -- Acknowledgments -- References -- Chapter 9: Practical Aspects of Adjusting Digital Cameras -- I. Introduction -- II. Measuring Gray-Level Information -- A. The Histogram -- B. The Line Scan -- C. Other Strategies -- III. Camera Settings -- A. Exposure Time -- B. Offset -- C. Gain -- IV. Contrast Stretching -- A. Setting the Exposure Time -- B. Adjusting Offset and Gain -- V. Camera Versus Image Display Controls -- Acknowledgments -- References -- Chapter 10: Cameras for Digital Microscopy -- I. Overview.
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II. Basic Principles -- A. Sources of Noise -- B. Quantum Efficiency -- C. Dynamic Range -- D. Image Integration -- E. CCD Architecture -- F. Scan Formats -- G. Binning -- H. Color -- I. Image-Intensified. CCD Camera -- J. Gating of the ICCD -- K. Electron-Bombarded CCD -- L. On-Chip Multiplication -- III. Application of CCD Cameras in Fluorescence Microscopy -- IV. Future Developments in Imaging Detectors -- References -- Chapter 11: A High-Resolution. Multimode Digital Microscope System -- I. Introduction -- II. Design Criteria -- A. Fluorescence Considerations -- B. Live Cell Considerations -- C. Phase Contrast and DIC Imaging -- D. The Need for Optical Sections -- III. Microscope Design -- A. Imaging Optics -- B. Epiillumination Optics -- C. Transillumination Optics -- D. Focus Control -- E. Vibration Isolation -- IV. Cooled CCD Camera -- A. Camera Features -- B. Quantum Efficiency -- C. Readout -- D. Linearity, Noise Sources, and the Advantages of Cooling -- E. Dark and Background Reference Image Subtraction -- F. Shading -- G. Signal-to-Noise -- V. Digital Imaging System -- A. Microscope Automation and Camera Control -- B. Other Useful Software and Devices -- VI. Example Applications -- A. DE-DIC -- B. Multimode Imaging of Anaphase In Vitro -- C. Fluorescence Photoactivation -- D. Microtubule and ER Dynamics in Migrating Tissue Cells -- E. GFP-Dynein-Labeled Microtubules in Yeast -- F. Immunofluorescent Specimens -- References -- Further Reading -- Chapter 12: Electronic Cameras for Low-Light Microscopy -- I. Introduction -- A. Low-Light Imaging in Biology -- B. The Importance of Proper Optical Imaging -- C. Detection of Low Light -- II. Parameters Characterizing Imaging Devices -- A. Sensitivity and Quantum Efficiency -- B. Camera Noise and the Signal-to-Noise Ratio -- C. Spatial Resolution -- D. Linearity and Uniformity -- E. Time Response.
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F. Dynamic Range -- G. Gain -- H. Frame Rate and Speed -- I. Resolution, Sensitivity, and Imaging Rate -- III. Specific Imaging Detectors and Features -- A. Video CCD Cameras -- B. Slow-Scan CCD Cameras -- C. Fast-Scan CCDs -- D. Intensified Cameras -- E. Internal Image Amplification Cameras -- F. Color Video Cameras -- G. Digital Still Cameras -- H. SIT Cameras -- I. CMOS Imagers -- J. Image Acquisition -- IV. Conclusions -- References -- Chapter 13: Camera Technologies for Low Light Imaging: Overview and Relative Advantages -- I. Overview -- A. Intensified CCD -- B. Cooled CCD -- C. Electron Bombardment CCD -- D. Intensified Electron Bombardment Camera -- E. Electron Multiplication CCD -- II. Sensitivity -- A. What Does "High Sensitivity" Mean? -- B. How Are Noise and Light Levels Related? -- III. Dynamic Range (DR) and Detectable Signal (DS) Change -- A. Definition of DR for a Camera -- B. Definition of DS Change -- C. Improving DR and DS Change -- D. Required Levels of SNR -- E. Sensitivity Comparison -- IV. Spatial Resolution Limits -- A. Intensified Camera -- B. Cooled CCD -- C. EB-CCD -- D. Intensified EB-CCD -- E. Electron Multiplication CCD -- V. Temporal Resolution -- VI. Geometric Distortion -- VII. Shading -- VIII. Usability -- A. Intensified Camera -- B. Cooled CCD -- C. Electron Bombardment CCD and Intensified Electron Bombardment -- D. EM-CCD -- IX. Advanced Technology -- A. High Sensitivity -- B. High Spatial Resolution -- C. High Temporal Resolution -- D. Convenience -- Acknowledgments -- References -- Chapter 14: Digital Manipulation of Brightfield and Fluorescence Images: Noise Reduction, Contrast Enhancement, and Feature Extraction -- I. Introduction -- II. Digitization of Images -- III. Using Gray Values to Quantify Intensity in the Microscope -- IV. Noise Reduction -- A. Temporal Averaging -- B. Spatial Methods.
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V. Contrast Enhancement.
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