Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes

TLDR

The study used giant unilamellar vesicles to measure diffusion of fluorescent lipid analogues in homogeneous and mixed lipid phases, revealing diffusion heterogeneity. FCS revealed single‑molecule lipid diffusion in membranes, showing deviations from two‑dimensional Brownian motion and suggesting anomalous subdiffusion or microdomain localization. Cytometry 36:176–182, 1999; © 1999 Wiley‑Liss, Inc.

Abstract

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases. Cytometry 36:176–182, 1999. © 1999 Wiley-Liss, Inc.