Water-soluble hormones and neurotransmitters are packaged in secretory vesicles and secreted into the extracellular medium by exocytosis, a process involving the fusion of the vesicle membrane with the cell membrane. Transport of the secretory vesicles to the cell s periphery, the maturation stages they undergo there to acquire fusion competence, and the factors controlling the fusion process itself (including the dynamics of the fusion pore) are important biological questions that are not fully understood. To elucidate secretory mechanisms at the single-vesicle level, currently only a few analytical methods exist, which can be grouped into electrical or optical recordings. The great advantage of electrical recordings (patch–clamp membrane capacitance and electrochemical amperometry) is their excellent time resolution (ca. tens of microseconds), which allows studies of the dynamics of the fusion pore itself. However, a major disadvantage is the fact that signals appear only after fusion has commenced; that is, the dynamics of the secretory vesicle itself or any labeled regulatory protein prior to the fusion event cannot be detected. In contrast, optical recordings allow secretory vesicles or regulatory proteins to be visualized and tracked prior to their fusion, yet generally they lack the time resolution required to follow the dynamics of the fusion pore (typical time resolution is ca. 100 ms). In addition, depending on the technique, secretion may be probed from different areas of a cell (top or bottom), which makes comparison of the results obtained by different approaches difficult. Because of their complementary nature, it would be a great advance if electrical and optical measurements could be made simultaneously from the same side of a cell at the singlevesicle level. This will enable a comprehensive and precise analysis of the whole exocytotic event, from predocking through fusion steps up to the dynamics of vesicular release. Herein, we report a device based on transparent indium tin oxide (ITO) electrodes, which allows simultaneous total internal reflection fluorescence microscopy (TIRFM) and amperometric measurements (Figure 1). As a proof of concept, the ability of our device in the coupled optical and electrochemical detections of exocytotic events is demonstrated using enterochromaffin BON cells. Amperometry is based on detection at a microelectrode surface positioned near the emitting cell of electroactive vesicular contents that are released into the extracellular medium. With very high temporal resolution and sensitivity, the flux of the vesicular content (released through an initial fusion pore that is only a few nanometers wide) corresponding to an exocytotic event appears as a current spike, which features (frequency, time length, area, magnitude) the dynamics of release from single vesicles. Generally, amperometry involves placing a large collecting electrode near the investigated cell. The whole cell active surface area is covered so the spatial localization of a particular exocytotic event cannot be achieved. Nevertheless, a few studies involving smaller microelectrodes or microelectrode arrays allowed amperometric signals from different releasing sites to be identified, but with a random positioning for the small microelectrode and a spatial resolution necessarily limited by the array dimensions, respectively. Coupling of amperometric and optical recordings would allow precise localization of exocytosis events in space and time. The most widely used optical approach to study exocytosis, TIRFM, is based on the total internal reflection of a laser beam at the glass/water interface, which creates an evanescent field in the aqueous medium whose characteristic decay length (ca. 100 nm) provides a high signal-to-noise ratio and an axial resolution of about 10 nm. When a vesicle fuses with the plasma membrane, its labeled contents are released toward the glass/water interface where the excitation [*] A. Meunier, Dr. R. Fulcrand, Dr. S. Arbault, Dr. M. Guille, Dr. F. Lema tre, Prof. C. Amatore D partement de Chimie, Ecole Normale Sup rieure UMR 8640 (CNRS-ENS-UPMC Univ Paris 06) 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-3863 E-mail: Christian.Amatore@ens.fr