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Proceedings Paper

Buffer controlled photoswitching microscopy using standard organic fluorophores
Author(s): Volker Buschmann; Samantha Fore; Sebastian van de Linde; Markus Sauer; Steve Wolter; Mike Heilemann; Felix Koberling; Rainer Erdmann
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Paper Abstract

The interest in super-resolution microscopy techniques has dramatically increased in the last years due to the unprecedented insight into cellular structure which has become possible [1]. In all widefield-based techniques, such as Stochastical Optical Reconstruction Microscopy (STORM) or Photo-activation localization microscopy (PALM), the dye-sensor-molecules are switched between a bright and a dark state. Many organic fluorophores exhibit intrinsic dark states with a lifetime that can be tuned by adjusting the level of oxidants and reductants in the buffer, thereby allowing to reversibly switch individual fluorophores between an on- and off-state [2]. This behavior is used in the dSTORM method. We exploited this redox-level adjusted photoswitching behaviour based on addition of millimolar amounts of reducing thiols for high-resolution imaging on a setup based on an inverse microscope coupled with ultrasensitive CCD camera detection. In order to quickly control the quality of the measurement, we used real-time computation of the subdiffraction-resolution image [3]. This greatly increases the applicability of the method, as image analysis times are greatly reduced.

Paper Details

Date Published: 23 February 2011
PDF: 7 pages
Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790510 (23 February 2011); doi: 10.1117/12.873383
Show Author Affiliations
Volker Buschmann, PicoQuant GmbH (Germany)
Samantha Fore, PicoQuant Photonics North America, Inc. (United States)
Sebastian van de Linde, Julius-Maximilians-Univ. Würzburg (Germany)
Markus Sauer, Julius-Maximilians-Univ. Würzburg (Germany)
Steve Wolter, Julius-Maximilians-Univ. Würzburg (Germany)
Mike Heilemann, Bielefeld Univ. (Germany)
Felix Koberling, PicoQuant GmbH (Germany)
Rainer Erdmann, PicoQuant GmbH (Germany)

Published in SPIE Proceedings Vol. 7905:
Single Molecule Spectroscopy and Imaging IV
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)

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