Two-photon-excitation fluorescence microscopy constitutes one of the most promising and fastest developing areas in biological and medical imaging at the optical-resolution level (Diaspro and Sheppard 2002). tte first application of two-photon fluorescence microscopy in biology was by Denket al. (1990) in 1990. tte benefits of two-photon excitation include improved background discrimination, reduced photobleaching of the fluorophores and minimal photodamage to living cell specimens (Denk et al. 1990; So et al. 1995,1996; Masters et al. 1999). In their application to membranes, the particular characteristics of multiphoton-excitation fluorescence microscopy allow the use of the UV-excited fluorescent probes discussed earlier in a microscope to fully combine and exploit two important pieces of information: (1) the fluorescence parameters that are sensitive to membrane lateral structure, such as phase-dependent emission shift, fluorescence lifetimes or polarization, as was done in the earlier studies in bulk, i.e., liposome solutions; (2) visual information (morphological and topological data). In two-photon-excitation microscopy, the fluorescent probes that are normally excited to the excited electronic state by UV photons can be excited by simultaneous absorption of two IR photons. As was previously demonstrated, the overall extent of photobleaching in this type of experiment is significant reduced compared with that in confocal fluorescence microscopy (Bagatolli and Gratton 2001; Bagatolli et al. 2003). Since their introduction, few two-photon-excitation fluorescence microscopy applications have been reported in order to explore the lateral structure of lipid membranes (Bagatolli and Gratton 2001; Quesada et al. 2001; Baumgart et al. 2003). Use of particular fluorescent probes and model membrane systems (ranging from single-component lipid membranes to cell membranes) allows important information regarding the microscopic scenario of li-pid-lipid and lipid-protein interactions to be achieved using UV-excited fluorescent probes and two-photon-excitation microscopy. Ms last fact offer a very consistent picture of relevant events such as lipid phase separation in lipid bilayers and cellular membranes (as discussed later), enzymatic reactions in membranes (Bagatolli et al. 2002; Sánchez et al. 2002) and insertion of peptides in lipid membranes (Fahsel et al. 2002; Janosch et al. 2004). Although other probes and methodologies can be applied to study membrane lateral heterogeneity, as discussed earlier, in the next sections the discussion will be limited primarily to results obtained with one of these probes, LAURDAN, in many different model system and native membranes using two-photon-excitation fluorescence microscopy.
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