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We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.

Original publication

DOI

10.1038/nmeth986

Type

Journal article

Journal

Nat Methods

Publication Date

01/2007

Volume

4

Pages

81 - 86

Keywords

Green Fluorescent Proteins, Microscopy, Fluorescence, Rhodamines, Sensitivity and Specificity