Table of Contents
Title Page
Copyright
List of Contributors
Preface to the Second Edition
What is This Book?
Why This Book?
Is This Book for You?
How Should You Read the Book?
What Updates Done to the First Edition?
Website of the Book
Personal Remarks on the History of This Book
Acknowledgments
Chapter 1: Introduction to Optics
1.1 A Short History of Theories about Light
1.2 Properties of Light Waves
1.3 Four Effects of Interference
1.4 Optical Elements
1.5 Optical Aberrations
References
Chapter 2: Principles of Light Microscopy
2.1 Introduction
2.2 Construction of Light Microscopes
2.3 Wave Optics and Resolution
2.4 Apertures, Pupils, and Telecentricity
2.5 Microscope Objectives
2.6 Contrast
2.7 Summary
Acknowledgments
References
Chapter 3: Fluorescence Microscopy
3.1 Contrast in Optical Microscopy
3.2 Physical Foundations of Fluorescence
3.3 Features of Fluorescence Microscopy
3.4 A Fluorescence Microscope
3.5 Types of Noise in a Digital Microscopy Image
3.6 Quantitative Fluorescence Microscopy
3.7 Limitations of Fluorescence Microscopy
3.8 Summary and Outlook
References
Recommended Internet Resources
Fluorescent Spectra Database
Chapter 4: Fluorescence Labeling
4.1 Introduction
4.2 Key Properties of Fluorescent Labels
4.3 Synthetic Fluorophores
4.4 Genetically Encoded Labels
4.5 Label Selection for Particular Applications
4.6 Summary
References
Chapter 5: Confocal Microscopy
5.1 Evolution and Limits of Conventional Widefield Microscopy
5.2 Theory of Confocal Microscopy
5.3 Applications of Confocal Microscopy
Acknowledgments
References
Chapter 6: Two-Photon Excitation Microscopy for Three-Dimensional Imaging of Living Intact Tissues
6.1 Introduction
6.2 What is Two-Photon Excitation?
6.3 How Does Two-Photon Excitation Microscopy Work in Practice?
6.4 Instrumentation
6.5 Limitations of Two-Photon Excitation Microscopy
6.6 When is 2PM the Best Option?
6.7 Applications of Two-Photon Microscopy
6.8 Other Nonlinear Microscopies
6.9 Future Outlook for 2PM
6.10 Summary
Acknowledgment
References
Chapter 7: Light Sheet Microscopy
7.1 Principle of Light Sheet Microscopy
7.2 Light Sheet Microscopy: Key Advantages
7.3 Construction and Working of a Light Sheet Microscope
7.4 Theory of Light Sheet Microscopy
7.5 Light Sheet Interaction with Tissue
7.6 3D Imaging
7.7 Multiview Imaging
7.8 Different Lens Configurations
7.9 Sample Mounting
7.10 Recent Advances in Light Sheet Microscopy
7.11 Outlook
7.12 Summary
References
Chapter 8: Localization-Based Super-Resolution Microscopy
8.1 Super-Resolution Microscopy: An Introduction
8.2 The Principle of Single-Molecule Localization Microscopy
8.3 Photoactivatable and Photoconvertible Probes
8.4 Intrinsically Photoswitchable Probes
8.5 Photoswitching of Organic Fluorophores by Chemical Reactions
8.6 Experimental Setup for Localization Microscopy
8.7 Optical Resolution and Imaging Artifacts
8.8 Fluorescence Labeling for Super-Resolution Microscopy
8.9 Measures for Improving Imaging Contrast
8.10 SMLM Software
8.11 Reference Structures for SMLM
8.12 Quantification of SMLM Data
8.13 Summary
References
Chapter 9: Super-Resolution Microscopy: Interference and Pattern Techniques
9.1 Introduction
9.2 Structured Illumination Microscopy (SIM)
9.3 Spatially Modulated Illumination (SMI) Microscopy
9.4 Application of Patterned Techniques
9.5 Conclusion
9.6 Summary
Acknowledgments
References
Chapter 10: STED Microscopy
10.1 Introduction
10.2 The Concepts behind STED Microscopy
10.3 Experimental Setup
10.4 Applications
10.5 Summary
References
Chapter 11: Fluorescence Photobleaching Techniques
11.1 Introduction
11.2 Basic Concepts and Procedures
11.3 Fluorescence Recovery after Photobleaching (FRAP)
11.4 Continuous Fluorescence Microphotolysis (CFM)
11.5 CLSM-Assisted Photobleaching Methods
11.6 Summary and Outlook
References
Chapter 12: Single-Molecule Microscopy in the Life Sciences
12.1 Encircling the Problem
12.2 What is the Unique Information?
12.3 Building a Single-Molecule Microscope
12.4 Analyzing Single-Molecule Signals: Position, Orientation, Color, and Brightness
12.5 Learning from Single-Molecule Signals
Acknowledgments
References
Chapter 13: Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging
13.1 General Introduction
13.2 Förster Resonance Energy Transfer
13.3 Measuring FRET
13.4 FLIM
13.5 Analysis and Pitfalls
13.6 Summary
References
Appendix A: What Exactly is a Digital Image?
A.1 Introduction
A.2 Digital Images as Matrices
A.3 Look-up Table
A.4 Intensity Histograms
A.5 Image Processing
A.6 Pitfalls
A.7 Summary
References
Appendix B: Practical Guide to Optical Alignment
B.1 How to Obtain a Widened Parallel Laser Beam?
B.2 Mirror Alignment
B.3 Lens Alignment
B.4 Autocollimation Telescope
B.5 Aligning a Single Lens Using a Laser Beam
B.6 How to Find the Focal Plane of a Lens?
B.7 How to Focus to the Back Focal Plane of an Objective Lens?
Index
End User License Agreement
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Guide
Table of Contents
Preface
Begin Reading