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As is the case with the vast majority of UV-excited fluorescent probes, confocal or epifluorescence microscopy experiments are difficult to perform with LAURDAN. As already mentioned, the extent of LAURDAN photobleaching under epifluorescence and confocal microscopy is severe, making it almost impossible to collect images on LAURDAN-labeled specimens for more than a few seconds (Bagatolli and Gratton 2001). A way to circumvent this problem is by using two-photon-excitation fluorescence microscopy. Because this fluorescent probe has very interesting spectroscopic and partition properties in membranes, it is possible to perform quantitative spectroscopic studies at each pixel and build an information image related to the sample at hand, be it a living cell, an extended surface polymer or a GUV. Researchers have been interested in this marriage of technologies and examples can be found in the early ratio imaging studies, fluorescence lifetime imaging techniques and fluorescence polarization imaging.

LAURDAN belongs to the family of polarity-sensitive fluorescent probes, first designed and synthesized by Gregorio Weber for the study of the phenomenon of dipolar relaxation of fluorophores in solvents, bound to proteins and associated with lipids (Weber and Farris 1979; Mcgregor and Weber 1986; Parasassi et al. 1986; La-sagna et al. 1996). When inserted in lipid membranes, LAURDAN displays unique characteristics compared with other fluorescent probes, namely, (1) LAURDAN shows a phase-dependent emission spectral shift, i.e., bluish in the ordered lipid phase and greenish in the disordered lipid phase (this effect is attributed to the reorientation of water molecules present at the lipid interface near LAURDAN's fluorescent moiety), (2) LAURDAN distributes equally into the ordered and disordered lipid phases, (3) the electronic transition moment of LAURDAN is aligned parallel to the hydrophobic lipid chains, allowing use of the photoselection effect to qualitatively discriminate between different lipid phases and (4) LAURDAN is negligibly soluble in water (Bagatolli and Gratton 2001; Bagatolli et al. 2003). tte homogeneous LAURDAN distribution in membranes displaying lateral heterogeneity together with the lipid phase-dependent emission spectral shift allows one to obtain lateral packing information directly from the fluorescence images. Ms fact offers a great advantage over the experimental techniques discussed before based on the probe partition to different lipid phases. A way to quantify the extent of water dipolar relaxation, that in turn is related to the phase state of the lipid membrane, is based on a useful relationship between the emission intensities obtained on the blue and the red side of LAURDAN's emission spectrum, ttis relationship, called generalized polarization (GP) (Parasassi and Gratton 1995; Parasassi et al. 1998), was defined by analogy to the fluorescence polarization function. In the GP function, the relative parallel and perpendicular orientations in the classical polarization function were

Generalized Polarization Laurdan

Fig. 9.1. LAURDAN emission propertiesand the LAURDAN generalized polarization (GP) function. The particular LAURDAN homogeneous distribution in membranes displaying phase coexistence (a) and the phase-sensitive emission shift (b) allows determination of the particular lipid phase coexistence scenario, c LAURDAN fluorescence intensity image of a giant unilamellar vesicle (GUV) composed of a 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/ 1,2-di-arachidoyl-sn-glycero-3-phosphocholine (DAPC) 1:1 molar binary mixture obtained using a blue band-passfilter (that selects the emission spectra in the blue region, i.e., the high-intensity area in the image corresponds to LAURDAN emission coming from the lipid gel phase), d LAURDAN GP image of the same phospholipid binary mixture. The high and low GP areas in the image correspond to gel and fluid phase, respectively. Notice the sensitivity of the GP function tothe lipid lateral organization.The barcorrespondsto 20 |im

Fig. 9.1. LAURDAN emission propertiesand the LAURDAN generalized polarization (GP) function. The particular LAURDAN homogeneous distribution in membranes displaying phase coexistence (a) and the phase-sensitive emission shift (b) allows determination of the particular lipid phase coexistence scenario, c LAURDAN fluorescence intensity image of a giant unilamellar vesicle (GUV) composed of a 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/ 1,2-di-arachidoyl-sn-glycero-3-phosphocholine (DAPC) 1:1 molar binary mixture obtained using a blue band-passfilter (that selects the emission spectra in the blue region, i.e., the high-intensity area in the image corresponds to LAURDAN emission coming from the lipid gel phase), d LAURDAN GP image of the same phospholipid binary mixture. The high and low GP areas in the image correspond to gel and fluid phase, respectively. Notice the sensitivity of the GP function tothe lipid lateral organization.The barcorrespondsto 20 |im substituted by the intensities at the blue and red edges of the emission spectrum (7b and 7r, respectively) using a given excitation wavelength. It is important to note that no polarizers are required in the experimental setup even though the name of this function contains the word polarization (Parasassi and Gratton 1995; Parasassi et al. 1998). tte GP parameter contains information about solvent dipolar relaxation processes which occur during the time that LAURDAN is in the excited state, and is related to water penetration in the lipid interfaces (Parasassi and Gratton 1995; Parasassi et al. 1998). tterefore, GP images can be constructed from the intensity images obtained with blue and green band-pass filters on the microscope, allowing further characterization of the phase state of the coexisting lipid domains (Bagatolli and Gratton 2001; Bagatolli et al. 2003).

Guvs Bagatolli

Fig.9.2. Photoselection effect on LAURDAN-labeled GUVs in the vesicle polar region (a). In the GUV's equatorial region the photoselection effect is abolished because of the parallel orientation of the LAURDAN transition moment with respect to the excitation light polarization plane (b), see text. These two situations (a, b) are sketched in three dimensions in c by taking into account the excitation light polarization plane (red areas containing the white arrows) that correspond with the scan region plane of the microscope. The lipid mixture in a corresponds to GUVs composed of a 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine/ceramide 5:1 molar mixture. The bar corresponds to 20 |am

Fig.9.2. Photoselection effect on LAURDAN-labeled GUVs in the vesicle polar region (a). In the GUV's equatorial region the photoselection effect is abolished because of the parallel orientation of the LAURDAN transition moment with respect to the excitation light polarization plane (b), see text. These two situations (a, b) are sketched in three dimensions in c by taking into account the excitation light polarization plane (red areas containing the white arrows) that correspond with the scan region plane of the microscope. The lipid mixture in a corresponds to GUVs composed of a 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine/ceramide 5:1 molar mixture. The bar corresponds to 20 |am

Figure 9.1 shows the homogeneous partition of LAURDAN between different lipid phases (Fig. 9.1a, c) and the particular emission spectra obtained in the gel and fluid phase regions (Fig. 9.1b). Discrimination of two different fluorescence intensity regions can be observed by using appropriate emission filters Fig. 9.1c. Additionally, computation of the GP function allows further characterization of the different lipid phases (Fig. 9.1d) (Bagatolli and Gratton 2001; Bagatolli et al. 2003). tte particular spherical shape of giant vesicles allows application of the photoselection effect to qualitatively distinguish between the different lipid phases, tte photoselection effect arises from the fact that only those fluorophores which have excitation transition moments aligned parallel, or nearly so, to the plane of polarization of the excitation light are excited. For example, with use of circularly polarized light as an excitation source, two different pictures can be observed in GUVs displaying gel/fluid phase coexistence at (1) the polar region of the vesicle (Fig. 9.2a) and (2) at the equatorial region of the vesicle (Fig. 9.2b). At the equatorial region of the vesicle the circularly polarized excitation light allows excitation with the same efficiency of all LAURDAN

molecules present in this region of the GUV (Bagatolli and Gratton 2001). In this case the transition moment of the probe is always parallel to the polarization plane, ttis last fact allows calculation of the GP images without the influence of the pho-toselection effect, as seen in Fig. 9.1d. On the other hand, at the polar region of the vesicle, only fluorescence coming from the fluid part of the bilayer can be observed (Fig. 9.2a), simply because a component of LAURDAN's transition moment is always parallel to the excitation polarization plane (because of the relatively low lipid order, i.e, the wobbling movement ofLAURDAN molecules). At the polar region of the vesicle no fluorescence intensity is observed in the gel phase areas because of the high lipid order (even though LAURDAN molecules are present in this region of the bilayer). tte message here is that the photoselection effect allows extraction of qualitative information about lipid phases directly from the intensity images.

tte photoselection effect can also be exploited to determine the orientation of the probe transition moment relative to the membrane plane (Bagatolli and Gratton 2000a; Bagatolli et al. 2000). tte fluorescence images obtained in the equatorial region of the vesicle using linearly polarized light as the excitation source will render particular characteristics depending on the probe orientation in the membrane. For example, in Fig. 9.3 the high-intensity areas in the fluorescence image are populated by fluorescent molecules with their transition moments oriented parallel to the direction of the polarized excitation light (because of the photoselection rule), tte immediate conclusion here is that the rhodamine-PE probe has its transition moment oriented 90° in the membrane with respect to that observed for LAURDAN. Additionally, LAURDAN GP images at the equatorial region of the GUV obtained with linearly polarized light provide information about coexistence of lipid domains with sizes below the resolution of the microscope (Parasassi et al. 1997; Bagatolli et al. 2003). For a detailed description of the use ofLAURDAN GP images to ascertain lipid domains below the resolution of the microscope the reader is encouraged to explore the works of Parasassi et al. 1997 and Bagatolli et al. 2003. To summarize, LAURDAN provides simultaneous information about the morphology and phase state of discrete regions in membranes directly from the fluorescence images, an advantage that is not obtained with other fluorescent probes.

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