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A Practical Guide to dSTORM: Super-Resolution Imaging with Standard Fluorescent Probes

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Far-Field Optical Nanoscopy

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 14))

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Abstract

This chapter provides a comprehensive overview of how reliable structural information can be obtained from super-resolution imaging based on stochastic photoswitching of organic fluorophores in fixed and living cells. Since single-molecule-based super-resolution imaging relies critically on the labeling density, the reversibility of photoswitching, and exact fitting of the center of mass of the measured point spread functions (PSFs) of isolated fluorophores, the controlled photoswitching of organic fluorophores with minimal photobleaching are discussed in detail, with particular focus on how they influence structural information extractable. Furthermore, the mechanism of reversible photoswitching of organic fluorophores in aqueous solvents in the presence of thiols is described. Finally, representative applications of direct stochastic optical reconstruction microscopy (dSTORM) are provided and consequences for live-cell super-resolution imaging with organic fluorophores with high spatiotemporal resolution is discussed.

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Authors and Affiliations

  1. Department of Biotechnology & Biophysics, Julius-Maximilans-University Würzburg, Am Hubland, 97074, Würzburg, Germany

    Markus Sauer

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  1. Markus Sauer
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Correspondence to Markus Sauer .

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Editors and Affiliations

  1. NanoBioSciences Institute of Physical and Theoretical Chemistry, TU Braunschweig, Braunschweig, Germany

    Philip Tinnefeld

  2. MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

    Christian Eggeling

  3. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Stefan W. Hell

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Sauer, M. (2012). A Practical Guide to dSTORM: Super-Resolution Imaging with Standard Fluorescent Probes. In: Tinnefeld, P., Eggeling, C., Hell, S. (eds) Far-Field Optical Nanoscopy. Springer Series on Fluorescence, vol 14. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2012_41

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