Introduction Vesicular exocytosis an intracellular membrane

Introduction Vesicular exocytosis, an intracellular membrane trafficking pathway, is a ubiquitous process for intercellular communication. It occurs when an intracellular vesicle fuses with the cell membrane and subsequently releases an infinitely minute number of chemical or biochemical messengers to the extracellular space within a brief fraction of time from milliseconds to seconds. This specific exocytotic process is found to be involved in plenty of normal and pathologic events in living 22(R)-hydroxy Cholesterol synthesis and its investigation thus attracts increasing research attention. In the past several decades, by recording the secretion course of vesicular contents, various optical [1], [2], [3], [4] and electrochemical methods [5], [6] have been developed to analyze this complicated biological process. Monoamine neurotransmitters are common signal molecules in cell communication and their unique feature of electroactivity makes it possible to track exocytotic process by electrochemical measurement [7], [8], [9]. Chronoamperometry stands out from a series of analytical methods as it is capable of quantifying the minute amount of important electroactive biomessengers contained within single vesicles. Indeed, amperometric detection of exocytosis at single cell level was firstly achieved with a carbon fiber microelectrode (CFE) by Wightman and his colleagues in the 1990s [10], [11]. Currently, serotonin and catecholamines such as dopamine, norepinephrine as well as epinephrine are the most commonly employed electroactive reporters for electrochemical test of cellular secretions via exocytosis. Additionally, the electroactivities of these neurotransmitters are all attributed to the oxidation of the phenol hydroxyl group. Recently, FFN102, a synthetic fluorescent false neurotransmitter possessing both one phenol hydroxyl group and a pyrone ring, was also introduced as an effective electrochemical probe for exocytosis tracking. The oxidizable analytes can be detected at femto-to zeptomole levels with temporal resolutions from microseconds to milliseconds. After three decades of development, single-cell amperometry has become an indispensable and the most widely spread tool for exocytosis investigation. Indeed, it benefits from its remarkable advantages including facility to implement, high sensitivity (1000 molecules per millisecond), as well as high temporal resolution (sub-millisecond time scale) [9], [12], [13]. More importantly, this technique is the only existing method capable of providing quantitative information of single-vesicle neurotransmitter release so far. In previous years, amperometric detection of cellular exocytosis was usually carried out with a single microelectrode, providing averaged information over the entire electrode surface area. This relative lack of spatial resolution thus became the major constraint of this technique. In order to make up this deficiency, various minimized micro/nanoelectrodes and multiple microelectrode arrays (MEAs) of subcellular size have been carefully designed to collect electrochemical information regarding to specific exocytotic zones on the surface of individual cells [14], [15]. In particular, the appearance of transparent/semi-transparent electrodes (ITO, diamond, thin gold film…) provides an opportunity to couple amperometry with other optical techniques which conveys spatially resolved information. The combination of electrochemistry and fluorescence microscopy offers synergetic advantages for exocytosis monitoring owing to their complementary characteristics. Specifically, time-resolved electrochemical signal is accompanied by in-situ fluorescent images, revealing quantitative information with both high temporal and spatial resolution. Additionally, the coupling methods promise the prospects of exploring the dynamics of individual secretory vesicles themselves or any labeled regulatory protein prior to the fusion event (vesicle translocation, docking and priming step), which cannot be achieved by solely using amperometry.