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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.

Original publication

DOI

10.1002/jemt.20505

Type

Journal article

Journal

Microsc Res Tech

Publication Date

12/2007

Volume

70

Pages

1003 - 1009

Keywords

Green Fluorescent Proteins, Photochemistry, Spectrometry, Fluorescence