The principal objective of this proposal is to develop and apply a methodology for determining the spatial distributions of membrane fluidity (and hence of lateral lipid segregation) and of other spectrofluorometrically-derived parameters, in individual cells, labelled with dynamic fluorescence probes. For example, the technique will be used to study the role of local heterogeneities in membrane fluidity in cellular processes, such as receptor- mediated endocytosis. In order to map fluidity or other structural and dynamic parameters, it is necessary to combine information from two (or more) fluorescence microscope images obtained under differ- ent spectral or polarization conditions. The instrumentation for this combinatorial imaging is designed around an inverted fluorescence microscope with filter wheels for exciting and emitted light, which will be equipped with a CCD-camera to obtain the required photo-sensitivity, resolution and image-stability. The system will employ an advanced image processing and analysis system which has been extensively tested in other (e.g. astronomical) applications. Among the practical aims which will be addressed in pursuit of these objectives are: 1) construction of the spectrofluorometric CCD microscope; 2) spectroscopic characterization of new dynamic fluorescence probes in vesicle systems and development of protocols for inserting them in cells; 3) two- and three-dimensional mapping of structural and dynamic parameters in sickle erythrocytes, epithelial cells and other systems.
