Trapped Brownian motion in single- and two-photon excitation fluorescence correlation experiments

We investigate possible trapping effects induced by the infrared radiation employed for two-photon fluorescence spectroscopy and microscopy on the diffusion of small fluorophores and draw a comparison to single-photon excitation. A first-order treatment of the effect of an optical trapping potential on the diffusion time and the number of molecules per excitation volume is also derived. By analyzing the fluorescence fluctuations arising from solutions of small dyes versus the excitation power and comparing them to the results with microspheres, we show that the bias on the molecular diffusion of,mail dyes is negligible when two-photon excitation is employed. In single-photon excitation, close to the resonance absorption of the dye, a small but detectable bias effect on the diffusion is found, in agreement with the study by Osborne et al.