Single-molecule Photoswitching and Localization

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doi:10.1071/CH10284

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Affiliated with RACI

Royal Australian
Chemical Institute

Single-molecule Photoswitching and Localization¹

Sebastian van de Linde ^A, Steve Wolter ^A and Markus Sauer ^A ^B

^A Biotechnology and Biophysics, Julius–Maximilians University Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany.
^B Corresponding author. Email: m.sauer@uni-wuerzburg.de

Australian Journal of Chemistry 64(5) 503-511 http://dx.doi.org/10.1071/CH10284
Submitted: 29 July 2010 Accepted: 4 November 2010 Published: 30 May 2011





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Abstract

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ~20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

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