Coupling electrochemistry and TIRF-microscopy with the fluorescent false neurotransmitter FFN102 supports the fluorescence signals during single vesicle exocytosis detection

Abstract

Applications of the Fluorescent False Neurotransmitter FFN102, an analog of biogenic neurotransmitters and a suitable probe for coupled amperometry and TIRFM (total internal reflexion fluorescence microscopy) investigations of exocytotic secretion, were considered here. The electroactivity of FFN102 was shown to very likely arise from the oxidation of its phenolic group through a CE (Chemical-Electrochemical) mechanism. Evidences that the aminoethyl group of FFN102 is the key recognition element by BON N13 cells were also provided. Amperometric measurements were then performed at the single cell level with carbon fiber electrode (CFE) or Indium Tin Oxide (ITO) surfaces. It proved the disparity of kinetic and quantitative parameters of FFN102-stained cells acquired either at cell top and bottom. Moreover, coupled analyses of FFN102 loaded vesicles allowed us to classify three types of optical signals that probably arise from secretion releases thanks to their concomitant detection with an electrochemical spike. Finally, preliminary benefits from the coupling involving FFN102 were reported in terms of origins of overlapped amperometric spikes or assignment of fluorescence extinctions to real exocytotic events.

Keywords: Coupling detection; Electrochemistry; Fluorescent false neurotransmitter; ITO; TIRF microscopy; Vesicular exocytosis.