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INTENSITY DEPENDENCE OF FLUOROPHORE PHOTOBLEACHING BY A STEPPED‐INTENSITY SLOW‐BLEACH EXPERIMENT
Author(s) -
Lanni Frederick,
Ware B. R.
Publication year - 1981
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1981.tb08998.x
Subject(s) - fluorophore , photobleaching , fluorescence , chemistry , intensity (physics) , bleach , light intensity , photochemistry , analytical chemistry (journal) , aqueous solution , fluorescein , optics , chromatography , physics , organic chemistry
— We describe a simple method for determination of the dependence of fluorophore photo‐bleaching on the intensity of the incident light. A sample of fluorophore is illuminated uniformly with an intensity which is sufficient only to bleach a small fraction of the fluorophore during a fluorescence lifetime period. The long‐term decay of fluorescence due to photobleaching is monitored before and after a stepped increase in the incident intensity. The order of the photobleaching reaction is determined by taking the ratio of the logarithms of the limiting values of the slope of the fluorescence bleaching curve and the absolute fluorescence change. The method is applied to fluorescein, bound to dextran or ovalbumin, in aqueous solution, and the order of the reaction in both cases is shown to be unity.