Digital Microscopy - 4th Edition - ISBN: 9780124077614, 9780124078925

Digital Microscopy, Volume 114

4th Edition

Editors: Greenfield Sluder David Wolf
eBook ISBN: 9780124078925
Hardcover ISBN: 9780124077614
Imprint: Academic Press
Published Date: 18th August 2013
Page Count: 672
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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


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


Details

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

About the Editors

Greenfield Sluder Editor

Affiliations and Expertise

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

David Wolf Editor

Affiliations and Expertise

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