A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution

Abstract

In this work, Fluorescent False Neurotransmitter 102 (FFN102), a synthesized analogue of biogenic neurotransmitters, was demonstrated to show both pH-dependent fluorescence and electroactivity. To study secretory behaviors at the single-vesicle level, FFN102 was employed as a new fluorescent/electroactive dual probe in a coupled technique (amperometry and total internal reflection fluorescence microscopy (TIRFM)). We used N13 cells, a stable clone of BON cells, to specifically accumulate FFN102 into their secretory vesicles, and then optical and electrochemical measurements of vesicular exocytosis were experimentally achieved by using indium tin oxide (ITO) transparent electrodes. Upon stimulation, FFN102 started to diffuse out from the acidic intravesicular microenvironment to the neutral extracellular space, leading to fluorescent emissions and to the electrochemical oxidation signals that were simultaneously collected from the ITO electrode surface. The correlation of fluorescence and amperometric signals resulting from the FFN102 probe allows real-time monitoring of single exocytotic events with both high spatial and temporal resolution. This work opens new possibilities in the investigation of exocytotic mechanisms.

Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements

“…For example, in patch-amperometry a carbon fiber electrode is positioned inside a patch pipette operated in cell-attached mode, capacitive measurements can be conducted in tandem with SCA to get information of number of molecules released and to monitor the change in membrane area [6]. Another example is the use of microfabricated ITO electrodes used in combination with total internal reflection (TIRF) microscopy that can be used to track fluorescently labeled vesicles during the later steps of exocytosis [7–10]. A wide range of labels are available from fluorescent proteins, membrane labels, and small molecules such as false fluorescent neurotransmitters (FFN) [10–14].…”

“…Another example is the use of microfabricated ITO electrodes used in combination with total internal reflection (TIRF) microscopy that can be used to track fluorescently labeled vesicles during the later steps of exocytosis [7–10]. A wide range of labels are available from fluorescent proteins, membrane labels, and small molecules such as false fluorescent neurotransmitters (FFN) [10–14]. …”

A High‐Affinity Fluorescent Sensor for Catecholamine: Application to Monitoring Norepinephrine Exocytosis

“…In order to validate that punctate drops in NS510 correspond to release of catecholamine from individual vesicles, experiments were performed using an amperometric ring microelectrode patterned underneath the cell being imaged . Amperometric microelectrodes immediately adjacent to release sites on the cell surface measure picoampere‐magnitude spikes of current as the catecholamine released from an individual vesicle is oxidized on the surface of the electrode . Figure presents three examples of amperometric spikes recorded synchronously in time with punctate changes in NS510 fluorescence indicated by the regions of interest.…”

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy

“…[8,9] Herein, fluorescence microscopy was coupled with aconventional three electrode electrochemical cell to investigate the potential dependent redox switch of organic fluorophores, thus demonstrating ap owerful tool to characterize the emissive properties of fluorescent and redox active molecules. This typically means ITO, [20][21][22][23][24][25] although studies of less common transparent materials are of interest. [15] This often means the fluorescent species are immobilized.…”

“…[18,19] Secondly,s urfaces that are both compatible with high-resolution light microscopy and are electroactive are required. This typically means ITO, [20][21][22][23][24][25] although studies of less common transparent materials are of interest. [9,26] Of the molecules that have been shown to undergo electrochemical switching of their fluorescence, improvements are needed in photostability [27] and fluorescence quantum yields.…”