Investigation of Diffusion in Structured Samples using Fluorescence Pair Cross Correlation

Abstract

Fluorescence Fluctuation Spectroscopy methods are powerful tools for studying diffusion both in vitro and in vivo. They are, however, limited to either homogeneous samples or to fit models that rely on generalized assumptions about the heterogeneity of the environment. Actual biological samples, however, often do not fulfill these conditions. The plasma membrane, for example, shows compartmentalization that changes dynamically [1]. This high level of complexity makes evaluation of results difficult.To better understand the effects of heterogeneities such as structures or obstacles on molecular diffusion, we monitored the diffusion of lipids in a simplified model system. We used a lipid bilayer on a nanostructured surface [2] as a simplified system to study the effect of compartmentalization on diffusion. Our samples consisted of hexagonally arranged compartments of lipid bilayers that are connected via nanometer gaps in between the metal structures. By calculating the spatial pair cross correlation functions [3] of a signal acquired while rapidly scanning over the sample, we could measure the time of diffusion of fluorescently labeled lipids between partitions. Since both diameter of the compartments and size of the gaps can be adjusted, this system allows us to study the influence of these parameters on diffusion. Furthermore, we performed Monte Carlo simulations of diffusion in similar structures. This way, we could test the effects of different interactions of the sample and the environment on the correlation functions.[1] T. Fujiwara et al., Journal of Cell Biology 2002, 157, 1071.[2] T. Lohmueller et al., Nano Letters 2012, 12, 1717.[3] M. Digman et al., Biophysical Journal 2009, 97, 665.