Cover

Main title

Copyright page

Contents

Preface

Abbreviations

1 Introduction

1.1 Classical Resolution Limit

1.1.1 Examples of Microscopic Imaging without Using Visible Light

1.1.2 Early Concepts of Enhanced Optical Resolution

1.1.3 Two-Photon and Near-Field Optical Microscopy

1.2 Methods to Circumvent the Classical Resolution Barrier in Fluorescence Microscopy

1.2.1 Interferometric Microscopy

1.3 Implementation of Super-Resolution Microscopy

1.4 Contrast

1.4.1 Multi-Color Imaging

1.5 Applications to the Study of Nuclear DNA

1.6 Other Applications

References

2 Physicochemical Background

2.1 Motivation

2.2 Labeling

2.2.1 Fluorophores

2.2.2 Methods of Labeling

2.2.3 Labeling Density

2.2.4 Binding

2.3 Fluorophore Transitions

2.3.1 Photobleaching

2.3.2 Photoswitching and Photon Yield

2.3.3 How to Achieve Switching, Blinking, Photostability, and High Photon Yield

2.3.4 Buffer Solutions for Combinations of Fluorophores

2.4 Samples

2.4.1 Optical Properties

2.4.2 Effects of Motion

2.4.3 Fixation

2.4.4 Diffusion

2.4.5 In vivo

References

3 Hardware and Software

3.1 Hardware Requirements

3.1.1 Collection of Fluorescence

3.1.2 Detectors

3.1.3 llumination

3.1.4 Adaptive Optics

3.1.5 Computer Technology

3.1.6 Overall System

3.2 Software

3.2.1 Feature Extraction

3.2.2 Error Correction

3.2.3 Visualization

3.2.4 Meta-Analysis

3.2.5 Confidence Analysis

3.3 Open Source and Best Practice

References

4 Structured Illumination and Image Scanning Microscopy

4.1 Axially Structured Illumination Microscopy

4.1.1 aSIM Setup

4.1.2 Principles of aSIM Size and Position Measurement

4.1.3 Requirements and Sample Preparation

4.1.4 Data Acquisition

4.1.5 Data Analysis and Visualization

4.1.6 Example Applications

4.2 Laterally Structured Illumination Microscopy

4.2.1 Principles of Lateral SIM

4.2.2 Implementation of Lateral SIM

4.2.3 Requirements and Sample Preparation

4.2.4 Data Acquisition

4.2.5 Data Analysis and Visualization

4.2.6 Example Applications

4.2.7 Imaging DNA Repair

4.3 Image Scanning Microscopy

4.3.1 Principles of Image Scanning Microscopy

4.3.2 Implementation of Image Scanning Microscopy

4.3.3 Requirements and Sample Preparation

4.3.4 Data Analysis and Visualization

4.3.5 Example Applications

4.3.6 Conclusion

4.4 Super-Resolution Using Rotating Coherent Scattering (ROCS) Microscopy

4.4.1 Principles of ROCS

4.4.2 ROCS Image Generation

4.4.3 Conclusion

References

5 Localization Microscopy

5.1 Principles of Localization Microscopy

5.2 PALM/STORM/fPALM/SPDM Approach

5.3 Implementation of SMLM

5.4 Principles of Three-Dimensional SMLM

5.5 Reduction of Out-of-Focus Light

5.6 How to Build a Three-Dimensional SMLM

5.7 High-Density Single-Emitter Microscopy Methods: SOFI, 3B, SHRImP, and Others

5.7.1 Independent Component Analysis

5.7.2 Single-Molecule High-Resolution Imaging with Photobleaching

5.7.3 Super-Resolution Optical Fluctuation Imaging (SOFI)

5.7.4 Bayesian Analysis of Blinking and Bleaching

5.7.5 Binding- and Activation-Assisted Separation

5.8 Approaches to Counting Molecules

5.8.1 Stepwise Photobleaching

5.8.2 Intensity Histogram Analysis

5.8.3 Multi-Color Colocalization

5.9 Requirements and Sample Preparation

5.9.1 Microtubule Staining for SPDMphymod and dSTORM

5.9.2 Imaging Buffer

5.9.3 Sampling

5.9.4 Counterstaining

5.9.5 Selection of Fluorophores

5.9.6 Cross-Talk

5.9.7 Illumination

5.10 Data Acquisition

5.11 Data Analysis

5.11.1 Effect of Threshold and Signal Detection

5.11.2 Extraction of Position, Photon Count, and Other Parameters

5.11.3 Excluding Imprecise Localizations

5.11.4 Assessing Image Resolution in SMLM

5.11.5 Available Software for SMLM Data Analysis

5.12 Troubleshooting

5.13 Meta Analysis Tailored for SMLM

5.13.1 Structure Averaging in Localization Microscopy

5.13.2 Pair Correlation Analysis

5.13.3 Analyzing Single-Molecule Trajectories

5.14 Example Applications

5.14.1 Multi-Color SMLM

5.14.2 Live-Cell SMLM

5.14.3 Structural Biology

5.14.4 Imaging in the Neurosciences

5.14.5 SMLM Spectroscopy

5.14.6 Example Applications to Chromatin Nanostructure

5.14.7 Combining Multiple Imaging Approaches

References

6 Stimulated Emission Depletion Microscopy

6.1 Principles of Stimulated Emission Depletion Microscopy

6.2 Implementation of STED

6.2.1 Pulsed STED (p-STED)

6.2.2 Continuous Wave STED

6.2.3 Gated cw STED

6.2.4 Protected STED

6.2.5 Generation of the STED Beam PSF

6.3 Fluorescent Probes

6.4 Dye Combinations for Dual-Color STED

6.5 Requirements and Sample Preparation

6.5.1 Base Instrument

6.5.2 Laser Light Sources

6.5.3 Choice of Detector

6.5.4 Obtaining a High-Quality PSF

6.5.5 Embedding Media

6.5.6 Sample Preparation Protocol

6.6 Data Acquisition

6.6.1 Adjusting for Cover Glass Thickness Using Correction Collar Ring

6.6.2 Pixel Size, Scan Speed, and Averaging

6.6.3 Adjust the Laser Power of the STED Depletion Beam

6.6.4 Increase the Signal from a STED Sample

6.7 Data Analysis and Visualization

6.7.1 Spectral Unmixing

6.7.2 Deconvolution

6.8 Example Applications

6.8.1 Multi-Color STED

6.8.2 Ultra-High-Resolution STED

6.8.3 In-Vivo STED

6.8.4 Deep Tissue Imaging

6.8.5 Imaging Fast Dynamics

6.8.6 Imaging Nuclear Chromatin

6.8.7 Imaging Techniques Combined with STED

6.9 Conclusion

References

7 Multi-Scale Imaging

7.1 Light-Sheet Fluorescence Microscopy

7.1.1 Principle of LSFM

7.1.2 Data Analysis and Visualization

7.1.3 Sample Preparation and Sample Mounting

7.1.4 Example Applications

7.1.5 Conclusion

7.2 Optical Projection Tomography

7.2.1 Principles of 3D Image Formation in OPT

7.2.2 OPT Setup

7.2.3 Requirements and Sample Preparations

7.2.4 Data Acquisition and Reconstruction

7.2.5 Example Applications

7.2.6 Conclusion

7.3 Expansion Microscopy and Sample Clearing

7.3.1 Principles of Expansion Microscopy

7.3.2 Implementation of Expansion Microscopy

7.3.3 Example Applications

7.3.4 Clearing

7.3.5 Conclusion

7.4 Alternative Approaches

References

8 Discussion

8.1 Future Challenges

8.1.1 Super-Resolution Microscopy Structural Analysis in Linear Excitation Mode

8.1.2 Quantification

8.1.3 In-Vivo Experiments Using STED and SMLM

8.1.4 Enhancement of Resolution

8.1.5 Multi-Color Experiments

8.1.6 Photophysics and the Development of Reporter Molecules

8.1.7 Novel Labeling Strategies

8.1.8 Fast and Accurate Software

8.1.9 Next-Generation Computing Hardware

8.1.10 Imaging of 3D Extended Objects

8.1.11 Super-Resolution Microscopy with a Large Field of View

8.1.12 Multi-Modal, Correlative Super-Resolution Imaging

8.1.13 Super-Resolution in Routine Applications

8.1.14 Super-Resolution Using Other Contrast Mechanisms

8.2 Commercialization of Super-Resolution Microscopes

8.3 Concluding Remarks

References

Index

EULA