Fluorescence techniques are powerful tools for studying biological processes. Therefore, the need for improved fluorescent probes used with these techniques continuously increases. The task of the present work was to characterize and to apply new fluorescent probes for biomembrane studies. For this purpose, two families of environment-sensitive probes have been considered. The first family is based on the 3-hydroxyflavone fluorophore (F2N12S, F46NS and F66NS). These dyes undergo an excited state intramolecular proton transfer reaction leading to their dual emission, highly sensitive to the environment. These probes allow the characterization of their environment properties through the ratio of intensities of their two bands (N*/T*) and present improved properties compared to F2N12S. The second family is based on the highly solvatochromic Nile Red fluorophore, exhibiting one emission band allowing a ratiometric measurement between its blue- and red-parts. We focused on one representative probe NR12S, which is a close structural analogue of F2N12S. The probe properties were characterized in lipid models, and in plasma membranes of living cells using a variety of fluorescence techniques (fluorescence spectroscopy, microscopy and flow cytometry). NR12S was then applied to study the lipid order in cell plasma membranes and its changes in response to several stimuli. Interestingly, this probes can evidence and monitor changes in the cell plasma membrane during apoptosis. Moreover, one novel application of the fluorescent membrane probe NR12S was the monitoring of lipid order in endocytic compartments during the maturation of endosomes. Thus, the described fluorescent membrane probes appear as powerful tools to determine the physico-chemical properties of the membrane and the relationship between lipid phase and physiological phenomena, such as apoptosis and endocytosis.
Ratiometric fluorescent membrane probes sensitive to lipid composition as tools for studies of lipid order, apoptosis and endocytosis