Fluorescent Correlation Spectroscopy and Raster Image Correlation Spectroscopy as a Tool to Measure Diffusion in the Human Epidermis

Abstract

Structural and dynamical characterization of skin tissue is vital for understanding the behavior of healthy and diseased skin tissue. Our objective is to develop protocols to measure the local diffusion of substances with different physical properties (for example amphiphilic or hydrophilic drugs) in the epidermis and dermis using fluorescent lipid analogs and hydrophobic dyes as model systems. As an example we use fluorescent labeled liposomes with a lipophilic dye in the bilayer and a hydrophilic dye inside. Using two color FCS (Fluorescent Correlation Spectroscopy) and two color RICS (Raster image correlation spectroscopy) we determine the diffusion and if intact liposomes penetrate the epidermis or if the burst before penetration. The experiments were preformed on a custom build multi-photon microscope[1]. Finally advantages and disadvantages of the different techniques for measuring diffusion in skin tissue are compared and discussed. [1] J. Brewer, J. Bernardino de la Serna, K. Wagner and L.A. Bagatolli. Multiphoton Excitation Fluorescence Microscopy in planar membrane systems , Biochim Biophys Acta,doi:10.1016/ j.bbamem.2010.02.024 (2010). [2] Susana A Sánchez and Enrico Gratton. Lipid-protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy. Acc Chem Res 38(6): 469-77, 2005. [3] Qiaoqiao Ruan, Melanie A Cheng,Moshe Levi, Enrico Gratton, and William W Mantulin. Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). Biophys J 87(2): 1260-7, 2004. [4] Claire M Brown, Rooshin B Dalal, B Hebert, Michelle A Digman, AlanR Horwitz, and Enrico Gratton. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229(1): 78-91, 2008.