Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

MARTÍN DODES TRAIAN; FRANCISCO LUIS GONZÁLEZ FLECHA; VALERIA LEVI

Buenos Aires

Workshop; 3rd South American Workshop on new trends in advanced fluorescence microscopy techniques; 2011

Our knowledge of the organization and function of biological membranes has changed from the original model of Singer and Nicholson. It is now well accepted that membrane components are not randomly organized but they assembly forming domains of different compositions, sizes and dynamics which are essential for the functionality of the membrane (1).In order to understand the physical basis of cell membrane organization and its relation to functionality, Giant Unilamellar Vesicles (GUVs) are a widely used system. GUVs sizes range between 10-50 μm and thus their curvatures are very similar to those of cells. Moreover, they can be easily imaged in microscopes allowing their study as individual liposomes. One of the key tools widely used for studying the fluidity of lipid membranes is the fluorescent probe 2-dimethylamino-6-lauroylnaphthalene (laurdan). This fluorescent molecule has exquisite photophysical properties that make it an almost ideal probe to sense changes in the lipid organization under different conditions (2, 3). Laurdan shows spectral sensitivity to the polarity of its environment, presenting a ~50-nm red shift of its emission spectra in polar solvents (3). Since loosely packed membranes present a higher penetration of water molecules, the emission spectra of laurdan in these membranes is red-shifted with respect to that observed in ordered membranes. One of the main drawbacks of laurdan is that it rapidly photobleaches under one photon excitation conditions determining that it can only be used in two photon excitation microscopy (4). This is a very important limitation since there are not too many laboratories with capability for two photon microscopy given the elevated cost of these microscopes.Recently Kim et al. (5) have synthesized a new fluorescent probe C-laurdan (6-dodecanoyl-2-[N-methyl-N-(carboxy- methyl)amino]naphthalene) designed for imaging and sensing lipid organization in membranes. C-laurdan fluorescence shows similar spectroscopic behavior to Laurdan and presents enhanced photostability under two-photon excitation and higher water solubility. In this work, we demonstrate that C-laurdan can be successfully used for imaging lipid organization in either artificial or natural membranes using a conventional confocal microscope setup. We believe that the use of this probe under one photon excitation will expand the possibilities of studying a wide variety of membrane-related biological processes.