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

作者：Guldbrand Stina^*; Kirejev Vladimir; Simonsson Carl; Goksor Mattias; Smedh Maria; Ericson Marica B

来源：European Journal of Pharmaceutics and Biopharmaceutics, 2013, 84(2): 430-436.

DOI：10.1016/j.ejpb.2012.10.001

• Two-photon fluorescence microscopy
• Fluorescence correlation spectroscopy
• Diffusion coefficient
• Human skin
• Skin penetration
• Allergens

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 %26lt; 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.

• 出版日期2013-6