Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging.
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 beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time 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 approximately 20 nm optical resolution.
van de Linde
Sebastian
S
Bielefeld Institute for Biophysics and Nanoscience, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.
Endesfelder
Ulrike
U
Mukherjee
Anindita
A
Schüttpelz
Mark
M
Wiebusch
Gerd
G
Wolter
Steve
S
Heilemann
Mike
M
Sauer
Markus
M