Fluorescence excitation volume in two-photon microscopy by autocorrelation spectroscopy and photon counting histogram

We compare two methods for the measurement of the excitation volume of an optical inverted microscope for two-photon excited fluorescence spectroscopy. The measure of the decay time of the fluorescence autocorrelation function gives the particles' diffusion time in the excitation volume, and from an a priori knowledge of the diffusion coefficient we can estimate the excitation volume. On the other hand, we can count the average number of particles in the excitation volume by analyzing the histogram of the fluorescence photon counting, and from the knowledge of the fluorophores' concentration we obtain the excitation volume. We show that the measurements of the excitation volume based on the analysis of the correlation function are very close, for a wide range of excitation volumes (0.1 fL < V-EXC < 6 fL), to those obtained with no a priori assumptions from the analysis of the photon counting histograms. Moreover, we suggest that the study of the relation between the average number of particles in the excitation volume and the nominal concentration can be a valuable tool in verifying the good alignment of the optical setup.