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Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively. This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe designed to mimic the well-known membrane probe LAURDAN's location and response in the lipid membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption and emission bands are red-shifted according to the polarity of the media. The photophysical behavior of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light (488 nm) as an excitation source. CAPRYDAA was also excitable by two-photon laser sources, making it easy to combine CAPRYDAA with either blue or red emission probes. In GUVs or cells, CAPRYDAA can discriminate the lipid phases and liquid-liquid phase heterogeneity. This new membrane probe shows the bathochromic properties of the PRODAN-based probes designed by Weber, overcoming the need for UV or two-photon excitation and facilitating the studies on the membrane properties using regular confocal microscopes.

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids.

A series of pyrroloquinolone photosensitizers bearing different halogen substituents (Cl, Br, I) on the heterocyclic framework was studied. These structures were readily prepared through a multi‐step synthetic sequence involving an oxidative protocol as an important step to access the quinolone framework. Spectroscopic characterizations and computational investigations were carried out to study the dyes before and after the oxidative step. Interestingly, the fluorescence emission was significantly reduced upon oxidation. In spite of a low photostability under UV light, the pyrroloquinolone photosensitizers proved effective to produce singlet oxygen. Higher singlet oxygen quantum yields were obtained with photosensitizers bearing halogen atoms with a higher atomic number. [ABSTRACT FROM AUTHOR]

The photophysics and photochemistry of a series of naphthoxazinones are studied by using steady-state, time-resolved spectroscopic techniques and product analysis. The presence of electron donors like amines has increased the photoconsumption quantum yield and has changed the product distribution, the primary product being a dihydronaphthoxazinone that has photoreacted by yielding ultimately an oxazoline derivative.

Research has been conducted on 2,3-dihydropyrazine derivatives. The reactions of singlet oxygen with these derivatives have been investigated, and the solvent effects have been analyzed in terms of both theoretical linear solvation energy and linear solvation energy relationships.

A series of pyrroloquinolone photosensitizers bearing different halogen substituents (Cl, Br, I) on the heterocyclic framework was studied. These structures were readily prepared through a multi‐step synthetic sequence involving an oxidative protocol as an important step to access the quinolone framework. Spectroscopic characterizations and computational investigations were carried out to study the dyes before and after the oxidative step. Interestingly, the fluorescence emission was significantly reduced upon oxidation. In spite of a low photostability under UV light, the pyrroloquinolone photosensitizers proved effective to produce singlet oxygen. Higher singlet oxygen quantum yields were obtained with photosensitizers bearing halogen atoms with a higher atomic number. [ABSTRACT FROM AUTHOR]

A series of pyrroloquinolone photosensitizers bearing different halogen substituents (Cl, Br, I) on the heterocyclic framework was studied. These structures were readily prepared through a multi-step synthetic sequence involving an oxidative protocol as an important step to access the quinolone framework. Spectroscopic characterizations and computational investigations were carried out to study the dyes before and after the oxidative step. Interestingly, the fluorescence emission was significantly reduced upon oxidation. In spite of a low photostability under UV light, the pyrroloquinolone photosensitizers proved effective to produce singlet oxygen. Higher singlet oxygen quantum yields were obtained with photosensitizers bearing halogen atoms with a higher atomic number., (© 2022 The Authors. Photochemistry and Photobiology published by Wiley Periodicals LLC on behalf of American Society for Photobiology.)