Super-Resolution Microscopy. A Practical Guide

  • ID: 4226242
  • Book
  • 408 Pages
  • John Wiley and Sons Ltd
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This unique book on super–resolution microscopy techniques presents comparative, in–depth analyses of the strengths and weaknesses of the individual approaches. It was written for non–experts who need to understand the principles of super–resolution or who wish to use recently commercialized instruments as well as for professionals who plan to realize novel microscopic devices. Explaining the practical requirements in terms of hardware, software and sample preparation, the book offers a wealth of hands–on tips and practical tricks to get a setup running, provides invaluable help and support for successful data acquisition and specific advice in the context of data analysis and visualization. Furthermore, it addresses a wide array of transdisciplinary fields of applications.

The author begins by outlining the joint efforts that have led to achieving super–resolution microscopy combining advances in single–molecule photo–physics, fluorophore design and fluorescent labeling, instrument design and software development. The following chapters depict and compare current main standard techniques such as structured illumination microscopy, single–molecule localization, stimulated emission depletion microscopy and multi–scale imaging including light–sheet and expansion microscopy. For each individual approach the experimental setups are introduced, the imaging protocols are provided and the various applications illustrated. The book concludes with a discussion of future challenges addressing issues of routine applications and further commercialization of the available methods.

Guiding users in how to make choices for the design of their own experiments from scratch to promising application, this one–stop resource is intended for researchers in the applied sciences, from chemistry to biology and medicine to physics and engineering.
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Preface

Abbreviations

INTRODUCTION

The Classical Resolution Limit

Methods to Circumvent the Classical Resolution Barrier in Fluorescence Microscopy

Implementation of Super–Resolution Microscopy (SRM)

Contrast

Applications to Study Nuclear DNA

Other Applications

PHYSICOCHEMICAL BACKGROUND

Motivation

Labeling

Transitions of the Fluorophores

Samples

HARD– AND SOFTWARE

Hardware Requirements

Software

Open Source and Best Practice

STRUCTURED ILLUMINATION AND IMAGE SCANNING MICROSCOPY

Axially Structured Illumination Microscopy (aSIM)

Laterally Structured Illumination Microscopy (SIM)

Image Scanning Microscopy

Super–Resolution Using Rotating Coherent Scattering (ROCS) Microscopy

LOCALIZATION MICROSCOPY

On the Principles of Localization Microscopy

PALM/STORM/fPALM/SPDM Approach

Implementation of SMLM

On the Principles of Three–Dimensional SMLM

Reduction of Out–of–Focus Light

How to Build a Three–Dimensional SMLM

High–Density Single Emitter Microscopy Methods: SOFI, 3B, SHRImP, etc.

Approaches Towards Counting Molecules

Requirements and Sample Preparation

Data Acquisition

Data Analysis

Troubleshooting

Meta Analysis Tailored for SMLM

Example Applications

STIMULATED EMISSION DEPLETION MICROSCOPY (STED)

On the Principles of Stimulated Emission Depletion Microscopy

Implementation of STED

Fluorescent Probes

Dye Combinations for Dual–Color STED

Requirements and Sample Preparation

Data Acquisition

Data Analysis and Visualization

Example Applications

Conclusion

MULTI–SCALE IMAGING

Light–Sheet Fluorescence Microscopy (LSFM)

Optical Projection Tomography (OPT)

Expansion Microscopy (ExM) and Sample Clearing

Alternative Approaches

DISCUSSION

Future Challenges

Commercialization of Super–Resolution Microscopes

Concluding Remarks

Index

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Udo J. Birk
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