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Digital Microscopy
- 4th Edition, Volume 114 - August 7, 2013
- Editors: Greenfield Sluder, David E. Wolf
- Language: English
- Hardback ISBN:9 7 8 - 0 - 1 2 - 4 0 7 7 6 1 - 4
- eBook ISBN:9 7 8 - 0 - 1 2 - 4 0 7 8 9 2 - 5
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 th… Read more
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Request a sales quoteThe 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
Cell and molecular biologists and researchers utilizing digitial microscopy techniques
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
- No. of pages: 672
- Language: English
- Edition: 4
- Volume: 114
- Published: August 7, 2013
- Imprint: Academic Press
- Hardback ISBN: 9780124077614
- eBook ISBN: 9780124078925
GS
Greenfield Sluder
Affiliations and expertise
University of Massachusetts Medical Center, Shrewsbury, U.S.A.DW
David E. Wolf
Affiliations and expertise
University of Massachusetts Medical Center, Shrewsbury, U.S.A.Read Digital Microscopy on ScienceDirect