Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes.

Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD (7-nitrobenz-2-oxa-1,3-diazole)-labeled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original fluorescence loss in electrobleaching (FLIE) assay. As expected, the reorganization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with fluorescence loss in photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar to that obtained by other methods reported in the literature. This versatile and selective bleaching procedure appears reliable to explore important biological and pharmacological issues.

A Fluorescent False Neurotransmitter as a Dual Electrofluorescent Probe for Secretory Cell Models.

A dual electrofluorescent probe (FFN42) belonging to the fluorescent false neurotransmitter family was rationally designed for investigating cell secretion. This probe, which comprises a coumarin core with one amino and two hydroxy groups, is very promising due to its electroactive and fluorescent properties. The optimal excitation and emission wavelengths (380 nm and 470 nm respectively) make this probe adapted for use in fluorescence microscopy. FFN42 has a quantum yield of 0.18, a molar absorption coefficient of 12000 M-1 cm-1 and pKa values of 5.4 and 6.7 for the hydroxy groups. The electroactivity of FFN42 was evidenced on carbon fiber and ITO electrodes at relatively low oxidation potentials (0.24 V and 0.45 V vs Ag/AgCl respectively). Epifluorescence observations showed that FFN42 accumulated into secretory vesicles of PC12 and N13 cells. Toxicity tests further revealed that FFN42 had no lethal effect on these cells. Amperometric data obtained on carbon fiber electrodes proved that the probe is released by N13 cells.