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Proceedings Article

Buffer controlled photoswitching microscopy using standard organic fluorophores

[+] Author Affiliations
Volker Buschmann, Felix Koberling, Rainer Erdmann

PicoQuant GmbH (Germany)

Samantha Fore

PicoQuant Photonics North America, Inc. (USA)

Sebastian van de Linde, Markus Sauer, Steve Wolter

Julius-Maximilians-Univ. Würzburg (Germany)

Mike Heilemann

Bielefeld Univ. (Germany)

Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790510 (February 22, 2011); doi:10.1117/12.873383
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From Conference Volume 7905

  • Single Molecule Spectroscopy and Imaging IV
  • Jörg Enderlein; Zygmunt K. Gryczynski; Rainer Erdmann
  • San Francisco, California, USA | January 22, 2011

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.

© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Citation

Volker Buschmann ; Samantha Fore ; Sebastian van de Linde ; Markus Sauer ; Steve Wolter, et al.
"Buffer controlled photoswitching microscopy using standard organic fluorophores", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790510 (February 22, 2011); doi:10.1117/12.873383; http://dx.doi.org/10.1117/12.873383


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