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Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin

Journal article
Authors Stina Guldbrand
Vladimir Kirejev
Carl Simonsson
Mattias Goksör
Maria Smedh
Marica B Ericson
Published in European Journal of Pharmaceutics and Biopharmaceutics
Volume 84
Issue 2
Pages 430-436
ISSN 0939-6411
Publication year 2013
Published at Department of Chemistry and Molecular Biology
Department of Physics (GU)
Core Facilities, Centre for Cellular Imaging
Pages 430-436
Language en
Keywords Two-photon fluorescence microscopy, Fluorescence correlation spectroscopy, Diffusion coefficient, living cells, microscopy, visualization, transport
Subject categories Pharmaceutical Sciences, Pharmacy


There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity. The measurements were performed at tissue depths in the range 0 and 20 pm, and the diffusion coefficients at skin depths 5 and 10 mu m were found to be significantly different (P < 0.05). Overall median values for the diffusion coefficients were found to be 4.0 x 10(-13) m(2)/s and 2.0 x 10(-13) m(2)/s for RB and RBITC, respectively. These values correspond to the diffusion of a hard sphere with a volume eight times larger for RBITC compared to RB. This indicates that the RBITC have bound to biomolecules in the skin, and the measured signal is obtained from the RBITC-biomolecule complexes, demonstrating the potential of the TPM-FCS method to track molecular interactions in an intricate biological matrix such as human skin.

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