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Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration |
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| Authors: | Eggeling C Hilbert M Bock H Ringemann C Hofmann M Stiel A C Andresen M Jakobs S Egner A Schönle A Hell S W |
| Affiliation: | 1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37070 G?ttingen, Germany;2. C. Eggeling and M. Hilbert contributed equally to this work |
| Abstract: | We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range. |
| Keywords: | fluorescence microscopy photoswitching reversibly switchable fluorescent proteins dronpa single‐molecule detection fluorescence correlation spectroscopy fluorescence intensity distribution analysis |
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