title: Monitoring triplet state dynamics with fluorescence correlation spectroscopy: bias and correction.
abstract: A marker's dark triplet state is of great importance in fluorescence microscopy: It serves as a means to switch off fluorescent markers and is thus the enabling element for several super-resolution methods. On the other hand, intersystem-crossing to the electronic dark triplet state strongly reduces the fluorescence yield in conventional fluorescence microscopy. The ability to determine the kinetic parameters of transitions into the triplet state is thus of great importance and because fluorescence correlation spectroscopy (FCS) can be applied without disturbing the system under study, it is one of the preferred methods to do so. However, conventional FCS observations of triplet dynamics suffer from bias due to the spatially inhomogeneous irradiance profile of the excitation laser. Herein, we present a novel method to correct this bias and verify it by analyzing both Monte Carlo simulated and experimental data of the organic dye Rhodamine 110 in aqueous solution for both continuous-wave and pulsed excitation. Importantly, our approach can be readily generalized to most other FCS experiments that determine intensity dependent kinetic parameters.
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schönle
authors.forename: Claas
authors.initials: C
authors.lastname: Von Middendorff
authors.forename: Christian
authors.initials: C
authors.lastname: Ringemann
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
mesh_terms: D005456:Fluorescent Dyes; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D009010:Monte Carlo Method; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes
keywords: fluorescence correlation spectroscopy; fluorescence microscopy; fluorescence saturation; intersystem-crossing; triplet