Fluorescence correlation spectroscopy with a doughnut-shaped excitation profile as a characterization tool in STED microscopy.

The resolution of stimulated emission depletion (STED) microscopes is ultimately limited by the quality of the doughnut-shaped illumination profile of the STED erase beam. We show here that in the focal plane this illumination profile is well approximated by an analytical expression - a difference of Gaussian functions, which tends towards a first order Laguerre-Gaussian profile in the case of a well aligned beam with a true zero-intensity central minimum. We further show that along the optical axis the maximum intensity profile is reasonably approximated by a Gaussian decay away from the focal plane. The result is a fully Gaussian analytical approximation of the three-dimensional point-spread function of STED erase beams. This allows the derivation of an analytical form for the autocorrelation function of the fluorescence generated by fluorophore diffusion through the STED depletion volume. We verified this form to be correct by performing fluorescence correlation spectroscopy (FCS) experiments in solutions of the dye Alexa Fluor 532. Since the quality of the illumination profile is reflected in the shape of the autocorrelation function, we propose that fluctuation analysis can be used as a tool to assess the quality of STED erase beams.