Digital Microscopy

Digital Microscopy

4th Edition - August 7, 2013
  • Editors: Greenfield Sluder, David Wolf
  • Hardcover ISBN: 9780124077614
  • eBook ISBN: 9780124078925

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Description

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.

Key Features

  • 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

Readership

Cell and molecular biologists and researchers utilizing digitial microscopy techniques

Table of Contents

  • Series Page

    Contributors

    Preface

    Chapter 1. Microscope Basics

    Introduction

    1.1 How Microscopes Work

    1.2 Objective Basics

    1.3 Mounting Video Cameras on the Microscope

    Reference

    Chapter 2. The Optics of Microscope Image Formation

    Introduction

    2.1 Physical Optics: The Superposition of Waves

    2.2 Huygens’ Principle

    2.3 Young’s Experiment: Two-Slit Interference

    2.4 Diffraction from a Single Slit

    2.5 The Airy Disk and the Issue of Microscope Resolution

    2.6 Fourier or Reciprocal Space: The Concept of Spatial Frequencies

    2.7 Resolution of the Microscope

    2.8 Resolution and Contrast

    Conclusions

    2.9 Appendix A

    2.10 Appendix B

    2.11 Appendix C

    Acknowledgments

    References

    Chapter 3. Proper Alignment of the Microscope

    3.1 Key Components and Image Locations of the Light Microscope

    3.2 Working the Microscope: Basic Setup

    3.3 Addendum

    Acknowledgments

    Chapter 4. Fundamentals of Fluorescence and Fluorescence Microscopy

    Introduction

    4.1 Light Absorption and Beer’s Law

    4.2 Atomic Fluorescence

    4.3 Organic Molecular Fluorescence

    4.4 Excited State Lifetime and Fluorescence Quantum Efficiency

    4.5 Excited State Saturation

    4.6 Nonradiative Decay Mechanisms

    4.7 Fluorescence Resonance Energy

    4.8 Fluorescence Depolarization

    4.9 Measuring Fluorescence in the Steady State

    4.10 Construction of a Monochromator

    4.11 Construction of a Photomultiplier Tube

    4.12 Measuring Fluorescence in the Time-Domain

    4.13 Filters for the Selection of Wavelength

    4.14 The Fluorescence Microscope

    4.15 The Power of Fluorescence Microscopy

    Acknowledgments

    References

    Chapter 5. Fluorescent Protein Applications in Microscopy

    5.1 The Identification of Green Fluorescent Protein

    5.2 Formation of the GFP Chromophore

    5.3 The Structure of GFP

    5.4 Mutagenesis to Alter the Properties of GFP

    5.5 Imaging FPs

    5.6 Applications of FP Imaging

    Conclusion

    References

    Chapter 6. Live-Cell Fluorescence Imaging

    Introduction

    6.1 Preparing a Specimen for Fluorescence Live-Cell Imaging

    6.2 Choice of Microscope

    6.3 Wide-Field Illumination of the Specimen

    6.4 Choosing the Best Objective Lens for Your Application

    6.5 Acquiring Digital Images Over Time

    6.6 ND Imaging

    6.7 Verifying Cell Health and Troubleshooting Sick Cells

    Conclusion

    Acknowledgments

    References

    Chapter 7. Practical Aspects of Adjusting Digital Cameras

    Introduction

    7.1 Measuring Gray-Level Information

    7.2 Camera Settings

    7.3 Contrast Stretching

    7.4 Camera Versus Image Display Controls

    Acknowledgments

    References

    Chapter 8. Cameras for Digital Microscopy

    8.1 Overview

    8.2 Basic Principles

    8.3 Application of CCD Cameras in Fluorescence Microscopy

    8.4 Future Developments in Imaging Detectors

    Further Reading

    Chapter 9. A High-Resolution Multimode Digital Microscope System

    Introduction

    9.1 Design Criteria

    9.2 Microscope Design

    9.3 Cooled CCD Camera

    9.4 Digital Imaging System

    9.5 Example Applications

    References

    Further Reading

    Chapter 10. Electronic Cameras for Low‐Light Microscopy

    Introduction

    10.1 Parameters Characterizing Imaging Devices

    10.2 Specific Imaging Detectors and Features

    Conclusions

    References

    Chapter 11. Camera Technologies for Low Light Imaging: Overview and Relative Advantages

    11.1 Overview

    11.2 CCD and sCMOS Technologies

    11.3 Low Light Camera Review

    11.4 Sensitivity

    11.5 Signal to Noise

    11.6 Comparing Camera Noise in Different Technologies

    11.7 DR and Detectable Signal Change

    11.8 Required Levels of Signal to Noise

    11.9 Sensitivity Comparison

    11.10 Spatial Resolution Considerations

    11.11 Temporal Resolution Considerations

    11.12 Geometric Distortion

    11.13 Shading

    11.14 Usability

    11.15 Advanced Technology Nutshell

    Acknowledgments

    Reference

    Chapter 12. Post-Processing for Statistical Image Analysis in Light Microscopy

    Introduction

    12.1 Digitization of Images

    12.2 Using Gray Values to Quantify Intensity in the Microscope

    12.3 Noise Reduction

    12.4 Contrast Enhancement

    12.5 Transforms, Convolutions, and Further Uses for Digital Masks

    12.6 Thresholding

    Conclusions

    References

    Chapter 13. 65,000 Shades of Grey: Use of Digital Image Files in Light Microscopy

    Introduction

    13.1 What is an Image File?

    13.2 Bit Depth

    13.3 File Formats

    13.4 Sampling and Spatial Resolution

    13.5 Color

    13.6 Converting RGB to CMYK

    13.7 Compression

    13.8 Video Files

    13.9 Video Codecs

    13.10 Choosing a Codec

    Conclusions

    Acknowledgments

    References

    Chapter 14. Quantitative Analysis of Digital Microscope Images

    14.1 So You Want to do Digital Imaging

    14.2 An Illustrative Example

    14.3 What is an Image?

    14.4 What Kind of Quantitative Information Do You Want?

    14.5 Quantitative Microscopy: A Tool Kit

    14.6 Exercise 1: A Simple Calibration Curve, Relative Scale

    14.7 Exercise 2: A Simple Calibration Curve, Absolute Scale

    14.8 Exercise 3: Precision in a Calibration Curve

    14.9 Standard Deviations, Standard Errors of the Mean, t‐Tests, and Other Confusions

    14.10 Dynamic Range, Do Not Waste it

    14.11 Signal‐to‐Noise Ratio, S/N, and Signal‐to‐Background Ratio, S/B

    14.12 Propagation of Error in Calculated Quantities

    14.13 Exercise 4: Error Propagation in Imaging

    14.14 Accuracy and Precision, Calibrating Against a Reference Standard

    14.15 Flatfield Correction

    14.16 Exercise 5: Flatfield Correction

    14.17 Applications Requiring Spatial Corrections

    14.18 Maximizing Resolution Before You Start

    14.19 Exercise 6: Converting Pixels to Microns

    14.20 Exercise 7: Imaging Warping

    14.21 Exercise 8: Two‐Color Coincidence

    14.22 Two‐Camera and Two‐Color Imaging

    14.23 Putting it All Together …

    14.24 Appendix A. Error Propagation: A Generalized Equation

    14.25 Appendix B. Image Translation and Rotation

    Acknowledgments

    References

    Chapter 15. Evaluating Optical Aberrations Using Fluorescent Microspheres: Methods, Analysis, and Corrective Actions

    Introduction

    15.1 Rationale

    15.2 Methods

    15.3 Discussion

    Summary

    Acknowledgments

    References

    Chapter 16. Ratio Imaging: Practical Considerations for Measuring Intracellular Ca2+ and pH in Living Cells

    Introduction

    16.1 Why Ratio Imaging?

    16.2 Properties of the Indicators BCECF and Fura-2

    16.3 Calibration of the Fluorescence Signal

    16.4 Components of an Imaging Workstation

    16.5 Experimental Chamber and Perfusion System: A Simple Approach

    Conclusion

    Acknowledgments

    References

    Chapter 17. Quantitative Fluorescence Microscopy and Image Deconvolution

    Introduction

    17.1 Quantitative Imaging of Biological Samples Using Fluorescence Microscopy

    17.2 Image Blurring in Biological Samples

    17.3 Applications for Image Deconvolution

    Concluding Remarks

    Acknowledgments

    References

    Chapter 18. Practical Aspects of Quantitative Confocal Microscopy

    Introduction

    18.1 Setting Up for Quantitative Imaging

    18.2 Correcting Nonuniformities (Flat Fielding)

    18.3 Limitations to Exact Quantitation

    18.4 Evaluating and Comparing Performance

    References

    Chapter 19. Theoretical Principles and Practical Considerations for Fluorescence Resonance Energy Transfer Microscopy

    Introduction

    19.1 Principles and Basic Methods of FRET

    19.2 FRET Microscopy

    Conclusions

    Acknowledgments

    References

    Chapter 20. Fluorescence Lifetime Imaging Microscopy for Quantitative Biological Imaging

    Introduction to Fluorescence Lifetime Imaging Microscopy

    20.1 Fluorophore Excited-State Lifetime: τ

    20.2 Methods for Creating Fluorescence Lifetime Maps

    20.3 FLIM Techniques for Quantitative Biological Imaging

    Summary

    Acknowledgment

    References

    Chapter 21. Fluorescence Correlation Spectroscopy: Molecular Complexing in Solution and in Living Cells

    Introduction

    21.1 Studying Biological Systems with FCS

    21.2 Designing and Building an FCS Instrument

    21.3 What Are the Current Commercial Sources of FCS?

    21.4 Summary

    Acknowledgments

    References

    Chapter 22. Breaking the Resolution Limit in Light Microscopy

    Introduction

    22.1 What Is Resolution?

    22.2 Methods Within the Combined Illumination and Detection Abbe Limit

    22.3 Methods Circumventing the Abbe Limit

    22.4 A Comment on Near Field Methods and Pendry’s Lens

    22.5 Key Points

    Acknowledgment

    References

    Further Reading

    Chapter 23. Circumventing Photodamage in Live-Cell Microscopy

    Introduction

    Conclusions

    References

    Chapter 24. A User’s Guide to Localization-Based Super-Resolution Fluorescence Imaging

    Introduction

    24.1 Fluorescent Probe Selection

    24.2 Sample Preparation

    24.3 Instrumentation

    24.4 Data Collection and Analysis

    Summary and Outlook

    Acknowledgments

    References

    Chapter 25. Quantitative Ratiometric Imaging of FRET-Biosensors in Living Cells

    Introduction

    25.1 Image Processing Methods

    25.2 Imaging Considerations and Caveats, Pitfalls

    Summary

    Acknowledgments

    References

    Chapter 26. Tip-Enhanced Raman Spectroscopy for the Base Interrogation of DNA

    Introduction and Motivations

    26.1 Materials

    26.2 Methods

    26.3 Discussion

    Summary

    References

    Index

    Volumes in Series

Product details

  • No. of pages: 672
  • Language: English
  • Copyright: © Academic Press 2013
  • Published: August 7, 2013
  • Imprint: Academic Press
  • Hardcover ISBN: 9780124077614
  • eBook ISBN: 9780124078925

About the Editors

Greenfield Sluder

Affiliations and Expertise

University of Massachusetts Medical Center, Shrewsbury, U.S.A.

David Wolf

Affiliations and Expertise

University of Massachusetts Medical Center, Shrewsbury, U.S.A.