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SYSM: Single Molecule Spectroscopy of Nanoobjects

SYSM 2: Single Molecule Spectroscopy of Nanoobjects II

SYSM 2.3: Invited Talk

Thursday, March 5, 2009, 15:20–16:00, VMP 8 HS

Photoswitching microscopy with subdiffraction optical resolution — •Markus Sauer, Mike Heilemann, and Sebastian van de Linde — Applied Laser Physics and Laser Spectroscopy, Bielefeld University, Universitaetsstr. 25, 33615 Bielefeld, Germany

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as ß-mercaptoethylamine and repopulation of the singlet ground state by reaction with molecular oxygen. Thus, the lifetime the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with ~ 20 nm optical resolution.