title: Enhancing fluorescence brightness: effect of reverse intersystem crossing studied by fluorescence fluctuation spectroscopy.
abstract: 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.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Ringemann
authors.forename: Andreas
authors.initials: A
authors.lastname: Schönle
authors.forename: Arnold
authors.initials: A
authors.lastname: Giske
authors.forename: Claas
authors.initials: C
authors.lastname: von Middendorff
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
mesh_terms: D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000528:radiation effects; D007700:Kinetics; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry* / Q000528:radiation effects; D008956:Models, Chemical; D010777:Photochemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins