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Enhancing Fluorescence Brightness: Effect of Reverse Intersystem Crossing Studied by Fluorescence Fluctuation Spectroscopy
Author(s) -
Ringemann Christian,
Schönle Andreas,
Giske Arnold,
von Middendorff Claas,
Hell Stefan W.,
Eggeling Christian
Publication year - 2008
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200700596
Subject(s) - intersystem crossing , photobleaching , fluorescence , photochemistry , chemistry , triplet state , photoexcitation , population , fluorescence spectroscopy , fluorescence cross correlation spectroscopy , fluorescence correlation spectroscopy , excited state , singlet state , atomic physics , optics , physics , demography , sociology
Experiments based on fluorescence detection are limited by the population of the fluorescence marker’s long‐lived dark triplet state, leading to pronounced photobleaching reactions and blinking which reduces the average fluorescence signal obtained per time interval. By irradiation with a second, red‐shifted laser line, we initiate reverse intersystem crossing (ReISC) which enhances the fluorescence signal of common fluorophores up to a factor of 14. The reverse intersystem crossing from the triplet state back to the singlet system is achieved by photoexcitation to higher‐excited triplet states, which are, however, prone to photobleaching. We gain insights into the competing pathways of ReISC and photobleaching. The relative efficiencies of these two pathways and the triplet lifetime determine the achievable fluorescence enhancement, which varies strongly with the choice of dye, excitation irradiance and wavelength, and with environmental conditions. The study of ReISC not only results in a better understanding of a fluorescent label’s photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter.