Fluorescence Microscopy E-Book

• Herausgeber: Wiley-VCH Verlag GmbH & Co. KGaA
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1032

Veröffentlichungsjahr: 2017

Beschreibung

While there are many publications on the topic written by experts for experts, this text is specifically designed to allow advanced students and researchers with no background in physics to comprehend novel fluorescence microscopy techniques.
This second edition features new chapters and a subsequent focus on super-resolution and single-molecule microscopy as well as an expanded introduction. Each chapter is written by a renowned expert in the field, and has been thoroughly revised to reflect the developments in recent years.

Leseprobe

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