FLUORESCENCE CORRELATION SPECTROSCOPY OF TRIPLET-STATES IN SOLUTION - A THEORETICAL AND EXPERIMENTAL-STUDY

A new approach for the study of singlet-triplet interactions of fluorophores in solution is presented, based on fluorescence correlation spectroscopy (FCS). The high excitation intensities used in FCS necessary for favorable S/N ratios can lead to significant intensity dependent distortions of the emission intensity distribution over the sample volume element from which fluorescence is collected and corresponding distortions of the fluorescence autocorrelation function. A theoretical outline of the photodynamical aspects of high excitation intensities in FCS is given, From this analysis, the rates of intersystem crossing and triplet decay as well as the excitation cross section of the fluorophore under investigation can be deduced. The triplet parameters of Rhodamine 6G (Rh6G) and Fluorescein isothiocyanate (FITC) have been determined in water and ethanol (Rh6G and FITC) and in methanol, propanol, and ethylene glycol (Rh6G). Also, the impact of potassium iodide and oxygen on the triplet parameters of Rh6G in water has been determined. The considerable spread in reference data and the relative simplicity of the method lead us to propose FCS as an appropriate method for the determination of triplet parameters of fluorophores in solution. Additionally, the comparatively high environmental sensitivity of these parameters indicates that triplet state monitoring by FCS can be used as a way of probing molecular microenvironments.