The Effect of Excited Fluorophore on Vesicle Fusion at the Surface of the Electrode

Abstract

Electrochemistry is a technique that can be used to detect the contents of neurotransmitter molecules in vesicles or liposomes by electrochemically oxidizing the content upon vesicle release. Previous work in quantifying the neurotransmitter content of single secretory vesicles has been performed using a technique called electrochemical cytometry. In our lab we have recently further developed this technique to become easier in performance and involves direct lysing of vesicles onto an amperometric electrode surface and without the need for a preceding separation step. Here we present how the electrochemical response from applying this new method to single adrenal chromaffin vesicles can be used to quantify vesicle content as well as study vesicle fusability as they impact a 33-um diameter disk-shaped carbon electrode. The frequency of the recorded amperometric spikes in each experiment has been used to probe the fusability of vesicle as a function of fluorofor concentration in the chromaffin vesicles membrane. Chromaffin granules were incubated with different concentration of fluorescent-labeled phospholipids before each experiment. In these experiments we used Fluorescent probe Rh-DOPE and or NBD-PS and probing vesicle fusion before and after subjection of samples to excitation wavelength of red (570nm) and blue (490nm) light respectively for each of the probes used. The data suggest a significant increase in the number and frequency of vesicles fusion onto the surface of the electrode when exciting the fluorophor of the granules incubated with fluorescently labeled phospholipid. Our finding show that the light itself or only labeled vesicle dose not change the frequency of vesicle fusion but the combination of theses two increase the chance of vesicle fusion. Therefore in this study we have shown that excited fluorescent-labeled phospholipids can change the membrane properties and facilitate vesicle fusability.