Thiazole orange (TO) exists mainly as a monomer in aqueous medium, where its fluorescence is negligibly small due to intramolecular movements. In the present study, it has been shown that in presence of giant unilamellar vesicles, produced from anionic lipid molecules, TO prefers to form H-dimer and H-aggregates at low lipid concentrations. The nonfluorescent form of TO (monomer) starts fluorescing in the aggregated or dimeric forms. At higher 1,2-dimyristoyl-sn -glycero-3-phospho-(1′-rac-glycerol) concentration, the TO aggregates disintegrate to the monomeric variants. This is principally due to generation of more surface of residence for the TO molecules. The dye molecules/aggregates reside on the outer surface as well as percolate inside the lipid vesicles toward the inner water pool due to the presence of anionic charges at the interface. We adopted fluorescence lifetime imaging to find out the heterogeneity in photophysics of the different forms of TO inside the lipid vesicles supported by fluorescence correlation spectroscopy to characterize the formation or disintegration of the TO aggregates.
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