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Photophysical Aspects of Single‐Molecule Detection by Two‐Photon Excitation with Consideration of Sequential Pulsed Illumination
Author(s) -
Niesner R.,
Roth W.,
Gericke KarlHeinz
Publication year - 2004
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.200300881
Subject(s) - photobleaching , intersystem crossing , excitation , autocorrelation , chemistry , fluorescence correlation spectroscopy , fluorescence , two photon excitation microscopy , molecular physics , excited state , atomic physics , optics , physics , statistics , mathematics , quantum mechanics , singlet state
An important goal in single molecule fluorescence correlation spectroscopy is the theoretical simulation of the fluorescence signal stemming from individual molecules and its autocorrelation function. The simulation approaches developed up to now are based exclusively on continuous‐wave (cw) illumination and consequently on cw‐excitation. However, this approximation is no longer valid in the case of two‐photon excitation, for which pulsed illumination is usually employed. We present a novel theoretical model for the simulation of the fluorescence signal of single molecules and its autocorrelation function with consideration of the time dependence of the excitation flux and thus of all illumination‐dependent photoprocesses: two‐photon excitation, induced emission and photobleaching. Further important characteristics of our approach are the consideration of the dependence of the photobleaching rate on illumination and the low intersystem‐crossing rates of the studied coumarins. Moreover, using our approach, we can predict quantitatively the effect of the laser pulse width on the fluorescence signal of a molecule, that is, the contributions of the photobleaching and saturation effects, and thus we can calculate the optimal laser pulse width. The theoretical autocorrelation functions were fitted to the experimental data, and we could ascertain a good agreement between the resulting and the expected parameters. The most important parameter is the photobleaching constant σ, the cross section of the transition S n ←S 1 , which characterises the photostability of the molecules independent of the experimental conditions. Its value is 1.7×10 −23 cm 2 for coumarin 153 and 5×10 −23 cm 2 for coumarin 314.