Buffer controlled photoswitching microscopy using standard organic fluorophores

Volker Buschmann; Samantha Fore; Sebastian van de Linde; Markus Sauer; Steve Wolter; Mike Heilemann; Felix Koberling; Rainer Erdmann

doi:10.1117/12.873383

23 February 2011 Buffer controlled photoswitching microscopy using standard organic fluorophores

Volker Buschmann, Samantha Fore, Sebastian van de Linde, Markus Sauer, Steve Wolter, Mike Heilemann, Felix Koberling, Rainer Erdmann

Author Affiliations +

Proceedings Volume 7905, Single Molecule Spectroscopy and Imaging IV; 790510 (2011) https://doi.org/10.1117/12.873383
Event: SPIE BiOS, 2011, San Francisco, California, United States

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.

Citation Download Citation

Volker Buschmann, Samantha Fore, Sebastian van de Linde, Markus Sauer, Steve Wolter, Mike Heilemann, Felix Koberling, and Rainer Erdmann "Buffer controlled photoswitching microscopy using standard organic fluorophores", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790510 (23 February 2011); https://doi.org/10.1117/12.873383

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