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

Abstract

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.

Combined scanning electrochemical and fluorescence microscopies using a tetrazine as a single redox and luminescent (electrofluorochromic) probe

“…The combination of electrochemical and fluorescence techniques has been the subject of growing interest among the scientific community from the beginning of the 1990s, starting with emission spectroelectrochemistry 1 and moving gradually towards more advanced configurations allowing spatially resolved measurements like fluorescence microscopy. 2 The topic was recently reviewed 3 and this combination of fluorescence and electrochemical techniques has been used to solve practical problems like the selective addressing of phospholipids in liposomes 4 or for imaging transient concentration profiles at microelectrode surfaces. 5 Among others, this combination has the great advantage of converting the electrochemical information, given by the current, into an optical signal, therefore improving the signal to noise ratio.…”

Electrochemical quenching of the fluorescence produced by NBD-labelled cell penetrating peptides: A contribution to the study of their internalization in large unilamellar vesicles

“…Indeed, NBD-tagged phospholipids have been found to undergo "leaflet-specific" electrochemical reduction in Giant Unilamellar Vesicles, i.e. only the external leaflet is electroactive, as confirmed with unsymmetrical vesicles [22].…”

“…Considering the redox nature of the reaction between dithionite and NBD, an electrochemical reduction could bring a significant benefit in several aspects [22]: i) It avoids adding exogenous chemical agents like dithionite ii) the heterogeneous nature of electron transfers achieved at solid electrodes imposes that the electrochemical reduction only affects species dissolved outside the vesicles or aggregated in the outer phospholipid leaflet of the vesicles iii) the counter-reaction (oxidation) can be separated in another compartment. Electrochemical activation/deactivation of fluorescent probes is not a new concept per se.…”

Electrochemical Fluorescence Switch of Organic Fluorescent or Fluorogenic Molecules

“…This work demonstrated how electrochemically-controlled fluorescence could unambiguously quantify the proportion of internalized peptides without interferences often observed with reducing agents. Interestingly, this work was further successfully extended to NBD-labelled phospholipids (P) in view of discriminating between outer and inner leaflets of vesicles [24]. Indeed, this distinction is impossible with usual fluorescence techniques such as FRAP (Fluorescence Recovery After Photobleaching) or FLIP (Fluorescence Loss In Photobleaching) due to limitations in optics and poor laser beam focusing resolution.…”

“…This allowed an estimation of the diffusion coefficient of phospholipids located on the outer-leaflet. By analogy with the FLIP technique (Fluorescence Loss In Photobleaching), the FLIE acronym was therefore introduced to qualify this powerful electrochemical quenching method (FLIE for: Fluorescence Loss In Electrobleaching) [24].…”