2-PHOTON FLUORESCENCE MICROSCOPY OF LAURDAN GENERALIZED POLARIZATION DOMAINS IN MODEL AND NATURAL MEMBRANES

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan, The GP function measures the relative water content of the membrane, In the present study we discuss images obtained with polarized laser e xcitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions, The GP distributi on in the gel-phase vesicles is relatively narrow, whereas the GP dist ribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is b road. Analysis of images obtained with polarized excitation of the liq uid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microsc ope resolution (similar to 200 nm radially and 600 nm axially), Vesicl es of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are p referentially excited by polarized light, have GP values lower than th e pure gel-phase domains. Cholesterol strongly modifies the domain mor phology. In the presence of 30 mol% cholesterol, the broad GP distribu tion of the DOPC/DPPC equimolar sample becomes narrower, The sample is still very heterogeneous, as demonstrated by the separations of GP di sjoined regions, which are the result of photoselection of regions of different lipid orientation, In intact red blood cells, microscopic re gions of different GP can be resolved, whereas in the renal cells GP d omains have dimensions smaller than the microscope resolution. Prepara tions of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different loca l orientation, or the domains may reflect a real heterogeneity of thes e membranes.