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Nanoimaging pp 131-151 | Cite as

Photoswitchable Fluorophores for Single-Molecule Localization Microscopy

  • Kieran Finan
  • Benjamin Flottmann
  • Mike Heilemann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 950)

Abstract

Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods—collectively referred to as “single-molecule localization microscopy” (SMLM)—are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.

Key to this strategy is the use of fluorophores that can be switched “on” and “off” in a controllable manner. Here we review the strengths and weaknesses of the wide variety of SMLM-compatible photoswitchable fluorophores and labeling strategies currently available. We also discuss their suitability for live-cell and multicolor imaging, as well as molecular counting.

Key words

PALM Photoactivated localization microscopy STORM dSTORM Stochastic optical reconstruction microscopy Super-resolution SMLM Single-molecule localization microscopy 

Notes

Acknowledgements

The authors are grateful to Sebastian Malkusch and Patrick Zessin for critical reading of the manuscript and to Prof. G.U. Nienhaus for providing spectral data for the fluorescent protein mEosFPthermo. This work was supported by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315262), the German Science Foundation (DFG, grant nr. HE 6166/2-1), and by contract research “Methoden für die Lebenswissenschaften” of the Baden-Württemberg Stiftung (grant nr. P-LS-SPII/11).

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Kieran Finan
    • 1
  • Benjamin Flottmann
    • 2
  • Mike Heilemann
    • 1
    • 2
  1. 1.Department of Biotechnology and BiophysicsJulius-Maximilians University WürzburgWürzburgGermany
  2. 2.Heidelberg Collaboratory for Image ProcessingUniversity of HeidelbergHeidelbergGermany

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