Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin

Abstract

Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 +/- 0.05 microm in the lateral direction, and 1.2 +/- 0.4 microm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 x 10(-8) cm(2)/s. The diffusion coefficient is not dependent on depths down to 40 microm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.