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.

Table of Contents:
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