Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes

Abstract

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 excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles 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 preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.

Differential Effect of Plant Lipids on Membrane Organization

“…The DOPC (T m ϭ Ϫ20°C)/DPPC (T m ϭ 41°C) lipid mixture, which has been widely used in the literature to characterize the physical properties of the membrane model system (11,14,83,108,109), exhibits, at temperatures below 45°C, an L o and L d phase coexistence (83). Performing our experiments at 22°C and minimizing temperature fluctuations, we actually favored a long time equilibrium formation of L o and L d phase separation, allowing us to focus our study on the structuring effect of plant lipid on L o and L d phase partitioning.…”

Phase separation is induced by phenothiazine derivatives in phospholipid/sphingomyelin/cholesterol mixtures containing low levels of cholesterol and sphingomyelin

“…In present work we describe the studies on the influence of three phenothiazine derivatives (trifluoperazine, chlorpromazine and thioridazine) on phase separation in mixtures containing low levels of cholesterol and sphingomyelin. The fluorescent indicator Laurdan was employed, whose generalized polarization is an ideal tool to follow the formation of domains in lipid systems [13,14]. It was found that all studied drugs induced phase separation, however the appearance of this effect depended on the phase state of the bilayer.…”

The Laurdan Spectral Phasor Method to Explore Membrane Micro-heterogeneity and Lipid Domains in Live Cells

“…These probes have equal and high affinity for every region of the membrane. Some examples of these fluorescent membrane markers are Laurdan (with its derivatives) [15][16][17][18][19][20][21], di-4-ANEPPDHQ [22,23], and other solvatochrome derivatives [24][25][26]. In this work we focus on the Laurdan probe and its capability to sense the polarity of its environment [27].…”