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We present a new method that combines fluorescence correlation spectroscopy(FCS) on the microsecond time scale with fluorescence antibunching measurementson the nanosecond time scale for measuring photophysical rate constants offluorescent molecules. The antibunching measurements allow us to quantify theaverage excitation rate of fluorescent molecules within the confocal detectionvolume of the FCS measurement setup. Knowledge of this value allows us then toquantify, in an absolute manner, the intersystem crossing rate and tripletstate lifetime from the microsecond temporal decay of the FCS curves. Wepresent a theoretical analysis of the method and estimate the maximum biascaused by the averaging of all quantities (excitation rate, photophysicalrates) over the confocal detection volume, and we show that this bias issmaller than 5\% in most cases. We apply the method for measuring thephotophysical rate constants of the widely used dyes Rhodamine~110 andATTO~655.

Measuring photophysical transition rates with fluorescence correlation spectroscopy and antibunching