We investigate the fluorescence emission characteristics of standard dye tetramethylrhodamine (TMR) in two-photon fluorescence correlation spectroscopy for different temporal and spectral properties of the femtosecond excitation pulses. After determining the second-order dispersion of our setup, including the microscope objective, a pulse stretcher was employed to control the temporal width at the location of the specimen. As expected, the fluorescence per molecule and therefore the signal-to-noise ratio of an FCS-measurement can be improved at constant average excitation power by altering either the temporal or spectral width of the excitation pulses. We found however, that the maximum achievable molecular brightness is largely independent of the temporal and spectral width in the regime analyzed. This observation confirms the current working hypothesis for two-photon fluorescence correlation spectroscopy that bleaching and saturation, and thus, the inherent properties of the dye system, are the dominant effects limiting the quality of measurements. As a practical consequence, elaborate optimization of temporal and spectral laser pulse width, e.g. by introducing pulse stretchers in the beam path, is less critical than previously expected.
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