Fluorescence microscopy : from principles to biological applications / edited by Ulrich Kubitscheck.

Format:

Book

Author/Creator:

Kubitscheck, Ulrich, editor.

Language:

English

Subjects (All):

Fluorescence microscopy.

Physical Description:

1 online resource (507 pages)

Edition:

2nd edition.

Place of Publication:

[Weinheim, Germany] : Wiley-VCH, 2017.

Summary:

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.

Contents:

Cover

Title Page

Copyright

Contents

List of Contributors

Preface

Chapter 1 Introduction to Optics

1.1 A Short History of Theories about Light

1.2 Properties of Light Waves

1.2.1 An Experiment on Interference

1.2.2 Physical Description of Light Waves

1.3 Four Effects of Interference

1.3.1 Diffraction

1.3.2 The Refractive Index

1.3.3 Refraction

1.3.4 Reflection

1.3.5 Light Waves and Light Rays

1.4 Optical Elements

1.4.1 Lenses

1.4.2 Metallic Mirrors

1.4.3 Dielectric Mirrors

1.4.4 Filters

1.4.5 Chromatic Reflectors

1.5 Optical Aberrations

References

Chapter 2 Principles of Light Microscopy

2.1 Introduction

2.2 Construction of Light Microscopes

2.2.1 Components of Light Microscopes

2.2.2 Imaging Path

2.2.3 Magnification

2.2.4 Angular and Numerical Aperture

2.2.5 Field of View

2.2.6 Illumination Beam Path

2.2.6.1 Critical and Köhler Illumination

2.2.6.2 Bright-Field and Epi-Illumination

2.3 Wave Optics and Resolution

2.3.1 Wave Optical Description of the Imaging Process

2.3.2 The Airy Pattern

2.3.3 Point Spread Function and Optical Transfer Function

2.3.4 Lateral and Axial Resolution

2.3.4.1 Lateral Resolution Using Incoherent Light Sources

2.3.4.2 Lateral Resolution of Coherent Light Sources

2.3.4.3 Axial Resolution

2.3.5 Magnification and Resolution

2.3.6 Depth of Field and Depth of Focus

2.3.7 Over- and Undersampling

2.4 Apertures, Pupils, and Telecentricity

2.5 Microscope Objectives

2.5.1 Objective Lens Design

2.5.2 Light Collection Efficiency and Image Brightness

2.5.3 Objective Lens Classes

2.5.4 Immersion Media

2.5.5 Special Applications

2.6 Contrast

2.6.1 Dark Field

2.6.2 Phase Contrast

2.6.2.1 Frits Zernike's Experiments

2.6.2.2 Setup of a Phase-Contrast Microscope.

2.6.2.3 Properties of Phase-Contrast Images

2.6.3 Interference Contrast

2.6.4 Advanced Topic: Differential Interference Contrast

2.6.4.1 Optical Setup of a DIC Microscope

2.6.4.2 Interpretation of DIC Images

2.6.4.3 Comparison between DIC and Phase Contrast

2.7 Summary

Acknowledgments

Chapter 3 Fluorescence Microscopy

3.1 Contrast in Optical Microscopy

3.2 Physical Foundations of Fluorescence

3.2.1 What is Fluorescence?

3.2.2 Fluorescence Excitation and Emission Spectra

3.3 Features of Fluorescence Microscopy

3.3.1 Image Contrast

3.3.2 Specificity of Fluorescence Labeling

3.3.3 Sensitivity of Detection

3.4 A Fluorescence Microscope

3.4.1 Principle of Operation

3.4.2 Sources of Exciting Light

3.4.3 Optical Filters in a Fluorescence Microscope

3.4.4 Electronic Filters

3.4.5 Photodetectors for Fluorescence Microscopy

3.4.6 CCD or Charge-Coupled Device

3.4.7 Intensified CCD (ICCD)

3.4.8 Electron-Multiplying Charge-Coupled Device (EMCCD)

3.4.9 CMOS

3.4.10 Scientific CMOS (sCMOS)

3.4.11 Features of CCD and CMOS Cameras

3.4.12 Choosing a Digital Camera for Fluorescence Microscopy

3.4.13 Photomultiplier Tube (PMT)

3.4.14 Avalanche Photodiode (APD)

3.5 Types of Noise in a Digital Microscopy Image

3.6 Quantitative Fluorescence Microscopy

3.6.1 Measurements of Fluorescence Intensity and Concentration of the Labeled Target

3.6.2 Ratiometric Measurements (Ca++, pH)

3.6.3 Measurements of Dimensions in 3D Fluorescence Microscopy

3.6.4 Measurements of Exciting Light Intensity

3.6.5 Technical Tips for Quantitative Fluorescence Microscopy

3.7 Limitations of Fluorescence Microscopy

3.7.1 Photobleaching

3.7.2 Reversible Photobleaching under Oxidizing or Reducing Conditions

3.7.3 Phototoxicity

3.7.4 Optical Resolution.

3.7.5 Misrepresentation of Small Objects

3.8 Summary and Outlook

Chapter 4 Fluorescence Labeling

4.1 Introduction

4.2 Key Properties of Fluorescent Labels

4.3 Synthetic Fluorophores

4.3.1 Organic Dyes

4.3.2 Fluorescent Nanoparticles

4.3.3 Conjugation Strategies for Synthetic Fluorophores

4.3.4 Non-natural Amino Acids

4.3.5 Bringing the Fluorophore to Its Target

4.4 Genetically Encoded Labels

4.4.1 Phycobiliproteins

4.4.2 GFP-Like Proteins

4.5 Label Selection for Particular Applications

4.5.1 FRET to Monitor Intramolecular Conformational Dynamics

4.5.2 Protein Expression in Cells

4.5.3 Fluorophores as Sensors Inside the Cell

4.5.4 Live-Cell Dynamics

4.5.5 Super-Resolution Imaging

4.6 Summary

Chapter 5 Confocal Microscopy

5.1 Evolution and Limits of Conventional Widefield Microscopy

5.2 Theory of Confocal Microscopy

5.2.1 Principle of Confocal Microscopy

5.2.2 Radial and Axial Resolution and the Impact of the Pinhole Size

5.2.3 Scanning Confocal Imaging

5.2.3.1 Stage Scanning

5.2.3.2 Laser Scanning

5.2.3.3 Spinning Disk Confocal Microscope

5.2.4 Confocal Deconvolution

5.3 Applications of Confocal Microscopy

5.3.1 Nonscanning Applications

5.3.1.1 Fluorescence Correlation Spectroscopy

5.3.1.2 Fluorescence Cross-Correlation Spectroscopy

5.3.1.3 Pulsed Interleaved Excitation

5.3.1.4 Burst Analysis with Multiparameter Fluorescence Detection

5.3.2 Scanning Applications beyond Imaging

5.3.2.1 Number and Brightness Analysis

5.3.2.2 Raster Image Correlation Spectroscopy

Chapter 6 Two-Photon Excitation Microscopy for Three-Dimensional Imaging of Living Intact Tissues

6.1 Introduction

6.2 What is Two-Photon Excitation?

6.2.1 Nonlinear Optics and 2PM.

6.2.2 History and Theory of 2PM

6.3 How Does Two-Photon Excitation Microscopy Work in Practice?

6.3.1 The Role of Light Absorption in 2PM

6.3.2 The Role of Light Scattering in 2PM

6.4 Instrumentation

6.4.1 Lasers for 2PM

6.4.2 Detection Strategies for 2PM

6.4.3 The Advantages of 2PM for Deep-Tissue Imaging

6.5 Limitations of Two-Photon Excitation Microscopy

6.5.1 Limits of Spatial Resolution in 2PM

6.5.2 Potential Sample Heating by the High Laser Powers in 2PM

6.5.3 Difficulties in Predicting and Measuring Two-Photon Excitation Spectra

6.5.4 Accelerated Photobleaching (and Associated Photodamage) in the Focal Plane

6.5.5 Expensive Lasers Create a Practical Limitation for Some Experiments

6.6 When is 2PM the Best Option?

6.6.1 Thick Specimen including In Vivo Imaging

6.6.2 Imaging Fluorophores with Excitation Peaks in the Ultraviolet (UV) Spectrum

6.6.3 Localized Photochemistry

6.7 Applications of Two-Photon Microscopy

6.7.1 Imaging UV-Excited Fluorophores, such as NADH for Metabolic Activity

6.7.2 Localized Photoactivation of "Caged" Compounds

6.7.3 Imaging Electrical Activity in Deep Tissue

6.7.4 Light Sheet Microscopy Using Two-Photon Excitation

6.7.5 Other Applications of 2PM

6.8 Other Nonlinear Microscopies

6.9 Future Outlook for 2PM

6.10 Summary

Acknowledgment

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

7.11.1 Big Data.

7.11.2 Smart Microscope: Imaging Concept of the Future

7.11.3 High-Throughput Imaging

7.12 Summary

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

8.8.1 Label Size versus Structural Resolution

8.8.2 Live-Cell Labeling

8.8.3 Click Chemistry

8.8.4 Three-Dimensional SMLM

8.8.5 Astigmatic Imaging

8.8.6 Biplane Imaging

8.8.7 Double Helix PSF

8.8.8 Interferometric Imaging

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

Chapter 9 Super-Resolution Microscopy: Interference and Pattern Techniques

9.1 Introduction

9.1.1 Review: The Resolution Limit

9.2 Structured Illumination Microscopy (SIM)

9.2.1 Image Generation in Structured Illumination Microscopy

9.2.2 Extracting the High-Resolution Information

9.2.3 Optical Sectioning by SIM

9.2.4 How the Illumination Pattern is Generated?

9.2.5 Mathematical Derivation of the Interference Pattern

9.2.6 Examples for SIM Setups

9.3 Spatially Modulated Illumination (SMI) Microscopy

9.3.1 Overview

9.3.2 SMI Setup

9.3.3 Excitation Light Distribution

9.3.4 Object Size Estimation with SMI Microscopy

9.4 Application of Patterned Techniques

9.5 Conclusion

9.6 Summary

Chapter 10 STED Microscopy

10.1 Introduction.

10.2 The Concepts behind STED Microscopy.

Notes:

Includes bibliographical references at the end of each chapters and index.

Description based on online resource; title from PDF title page (ebrary, viewed April 20, 2017).

Other Format:

print book

ISBN:

9783527687749

3527687742

9781523115303

1523115300

9783527687725

3527687726

9783527687732

3527687734

OCLC:

982011816