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

Journal article
Authors Stina Guldbrand
Carl Simonsson
Mattias Goksör
Maria Smedh
Marica B Ericson
Published in Optics Express
Volume 18
Issue 15
Pages 15289-15302
ISSN 1094-4087
Publication year 2010
Published at Department of Chemistry
Department of Physics (GU)
Pages 15289-15302
Language en
Links dx.doi.org/10.1364/OE.18.015289
Subject categories Optics

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 μm in the lateral direction, and 1.2 ± 0.4 μm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 × 10 −8 cm2/s. The diffusion coefficient is not dependent on depths down to 40 μm. 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.

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