Introduction

tte idea that lipids are simply randomly organized building blocks of membranes that form diffusion barriers between cytoplasm and the outside world has significantly changed since the discovery of the coexistence of stable lipid domains in lipid bilayers, approximately 3 decades ago (Gebhart et al. 1977). Conversely, the consequences of the nonrandom lateral organization of lipid membranes have not been acknowledged, particularly from the biology field, until recently when the raft hypothesis was postulated (Simons and Ikonen 1997; Brown and London 1998; Edi-din 2003). At present, however, the connection between membrane lateral structure and membrane function remains obscure. Still, there are simple questions regarding lipids and membranes that still need to be answered. For example, why do cells membranes contain thousands of different molecular lipid species? Why do the molar fractions of these species vary among different membranes? Is there a coherent code still hidden and waiting to be discovered? As Hilgemann (2003) pointed out in his article "Getting ready for the decade of the lipids" ".. .why not speculate that (phospho)lipids and their metabolites will soon be the subject of an information explosion, similar to that presently occurring for genes and proteins?"

In the last 30 years, there has been extensive research to elucidate the coexistence of lipid domains in membranous systems (mainly liposomes but also cell membranes) using an array of experimental techniques such as fluorescence spectroscopy, differential scanning calorimetry, IR spectroscopy, electron paramagnetic resonance, NMR to mention a few (Lee 1975; Lentz et al. 1976; Mabrey and Sturtevant 1976; van Dijjck et al. 1977; Arnold et al. 1981; Blume et al. 1982; Cafrey and Hing 1987; Shimshick and McConnell 1973; Maggio 1985; Maggio et al. 1986; Bagatolli et al. 1997; Vaz et al. 1989, 1990; Bultmann et al. 1991; Almeida et al. 1992; Parasassi et al. 1993; Schram et al. 1996), including theoretical treatments using computer simulations (Ipsen and Mouritsen 1988; j0rgensen and Mouritsen 1995). In general, the experimental techniques just indicated produce mean parameters on the basis of data collected from bulk solution of many liposomes (or cells) and lack information about lipid lateral organization at the level of single vesicles, a quality that can be provided by microscopy techniques, in particular fluorescence microscopy, tte main advantage in using fluorescence microscopy related techniques in membranes over the traditional experimental approaches is clear: the sensitivity and flexibility of a microscope with the addition of fluorescence spectroscopy allows the collection of spatially resolved information. Ultimately this information bridges membrane mor-

Springer Series in Biophysics J.L.R. Arrondo and A. Alonso Advanced Techniques in Biophysics © Springer-Verlag Berlin Heidelberg 2006

phology with dynamical and structural information obtained at a molecular level using fluorescence spectroscopy (such as lipid mobility and hydration), tte additional "visual" information obtained in these experiments has generated new data in the membrane field (in particular for bilayer membrane systems) that have important implications in understanding membrane lateral structure, tte aim of this report is to summarize the overall impact of the newly obtained results using fluorescence microscopy in the membrane field from model systems of different composition to native membranes. Also the most relevant aspects of the experimental methodology will be briefly discussed.

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