title: Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin.
abstract: 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 μm, and the diffusion coefficients at skin depths 5 and 10 μm were found to be significantly different (P<0.05). Overall median values for the diffusion coefficients were found to be 4.0×10(-13) m(2)/s and 2.0×10(-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.
authors.affiliation: Department of Physics, University of Gothenburg, Gothenburg, Sweden. stina.guldbrand@physics.gu.se
authors.forename: Stina
authors.initials: S
authors.lastname: Guldbrand
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kirejev
authors.forename: Carl
authors.initials: C
authors.lastname: Simonsson
authors.forename: Mattias
authors.initials: M
authors.lastname: Goksör
authors.forename: Maria
authors.initials: M
authors.lastname: Smedh
authors.forename: Marica B
authors.initials: MB
authors.lastname: Ericson
mesh_terms: D000287:Administration, Topical; D000485:Allergens / Q000737:chemistry; D002626:Chemistry, Pharmaceutical / Q000295:instrumentation / Q000379:methods*; D004058:Diffusion; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D012235:Rhodamines / Q000737:chemistry*; D012867:Skin / Q000187:drug effects / Q000473:pathology*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000485:Allergens; D005456:Fluorescent Dyes; D012235:Rhodamines; C027755:rhodamine isothiocyanate; C029773:rhodamine B