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Bio-Optical Investigations
Dynamic Spectroscopy

Giant Unilamellar Vesicles
 
Probing lateral dynamics and phase behavior in model and cell membranes by means of fluorescence correlation spectroscopy (FCS) and confocal microscopy
 

The phase behavior of biological membranes is closely related to several functional processes of a living cell, such as signaling, protein sorting, membrane fusion and membrane fission. Numerous studies provide evidence for the fact that cell membranes are laterally inhomogeneous, and that a controlled lateral (co-)segregation of membrane components is essential for the above-mentioned processes. The membrane fluid-mosaic model accordingly has to be modified. The membrane raft model has become a popular description of heterogeneous biological membranes: liquid-ordered lipid domains, enriched in cholesterol and sphingomyelin, function as rafts and signaling platforms, in a separated, liquid-disordered membrane phase. The underlying physicochemical principles of domain formation and clustering, and the properties of coexisting membrane domains are currently not well understood. We apply confocal microscopy and fluorescence correlation spectroscopy to determine phase behavior, the nature of phase transitions and the properties of coexisting phases, both in model membrane systems and in biological membranes. This project is performed in cooperation with Prof. G.W. Feigenson, Cornell University.
 
 
 
 
Biological membranes naturally consist of numerous components, which complicates the determination of their phase behavior. The above phase diagram (Feigenson et al., Biophys. J. 80, 2775 (2001)) is based on a ternary lipid mixture, which is meant to serve as a model for the lipid composition of the outer leaflet of the plasma membrane.
 
   
 
Confocal microscopy with differentially participating fluorescence lipid probes is an excellent way of studying microscopically resolvable lipid domains, in freely suspended membranes of giant unilamellar vesicles (GUVs).

FCS was first applied to the study of lateral diffusion in model membranes by the Webb group in 1978. While photobleaching recovery has become the most widely used methodology for analyzing lateral diffusion of membrane components in both biological and model membranes, modern FCS offers several advantages. First, FCS allows - after careful correction for background - the determination of the average number of molecules in the focal volume, thus enabling the determination of partition coefficients of membrane dyes into separated phases. Second, by FCS one can perform multicolor, simultaneous measurements of the diffusion behavior of differentially participating lipid dyes. Third, unlike photobleaching, FCS does not require a quasi-infinite reservoir of unbleached molecules for the determination of accurate diffusion kinetics. Therefore, FCS allows the measurement of diffusion coefficients in relatively small membrane domains. Fourth, FCS can be used to determine spatial correlation lengths of concentration fluctuations. Model membranes in the form of GUVs can be used with advantage for the studies described above, since artifactual membrane/substrate interactions (as in supported lipid membranes) or averaging over several bilayers (as in multi-bilayer stacks) are absent. Particularly interesting, and biologically highly relevant, are model membrane mixtures, which show a macroscopic phase separation into liquid-ordered and liquid-disordered phases, with circular domain shapes dominated by line tension.
 
 
 
 
 

Selected publications:

Webb, W. W. (2001). Fluorescence Correlation Spectroscopy: Genesis, evolution, maturation and prognosis. Fluorescence Correlation Spectroscopy Theory and Applications. R. Rigler and E. S. Elson. Berlin, Springer-Verlag: 305-330.

Schwille, P., J. Korlach, et al. (1999). "Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes." Cytometry 36(3): 176-182.

Korlach J., Schwille P., et al. (1999). "Characterization of Lipid Bilayer Phases by Confocal Microscopy and Fluorescence Correlation Spectroscopy." PNAS USA 96: 8461 - 8466.

Fahey, P. F. and W. W. Webb (1978). "Lateral Diffusion in Phospholipid Bilayer Membranes and Multilamellar Liquid-Crystals." Biochemistry 17(15): 3046-3053.

Magde, D., W. W. Webb, et al. (1972). "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy." Physical Review Letters 29(11): 705-708.