We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 μm diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.

The mechanism of mammalian vesicle rupture onto the surface of a polarized carbon fiber microelectrode during electrochemical vesicle cytometry is investigated. It appears that following adsorption to the surface of the polarized electrode, electroporation leads to the formation of a pore at the interface between a vesicle and the electrode and this is shown to be potential dependent. The chemical cargo is then released through this pore to be oxidized at the electrode surface. This makes it possible to quantify the contents as it restricts diffusion away from the electrode and coulometric oxidation takes place. Using a bottom up approach, lipid-only transmitter-loaded liposomes were used to mimic native vesicles and the rupture events occurred much faster in comparison with native vesicles. Liposomes with added peptide in the membrane result in rupture events with a lower duration than that of liposomes and faster in comparison to native vesicles. Diffusional models have been developed and suggest that the trend in pore size is dependent on soft nanoparticle size and diffusion of the content in the nanometer vesicle. In addition, it appears that proteins form a barrier for the membrane to reach the electrode and need to move out of the way to allow close contact and electroporation. The protein dense core in vesicles matrixes is also important in the dynamics of the events in that it significantly slows diffusion through the vesicle.

We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size. KEYWORDS: nanoimaging, NanoSIMS, vesicle content, electrochemistry, nanocompartments C hemical communication in eukaryotic cells relies heavily on loading and trafficking of secretory vesicles to the plasma membrane. Upon stimulation, the loaded, docked secretory vesicles fuse with the plasma membrane and discharge their neurotransmitter cargo into the extracellular space. This process, called exocytosis, is fundamental and highly regulated. 1,2 Regulation of exocytosis has been widely studied, and the understanding of its mechanism is still under debate. Several mechanisms have been reported such as all-or-nothing release during which vesicles completely fuse with the plasma membrane and release their total content. 3 Contrary to the all-or-nothing mode, partial release mechanisms have been proposed, suggesting the transient opening of the fusion pore between the cell membrane and secretory vesicle followed by closing again. 4 In addition to the release event, exocytosis is also regulated through vesicle loading. In general, this process involves a neurotransmitter transporter, an integral vesicle membrane protein that uses the pH gradient across the vesicle membrane, to drive uptake of neurotransmitters into the vesicle. 5−8 Defining the vesicle storage mechanisms would be an important piece of the exocytosis regulation puzzle. 5,9,10 Several cell types have been employed for loading studies among which neuroendocrine cells have had an important role as a model system. They possess two types of secretory vesicles, synaptic-like microvesicles (SLMVs) and so-called large densecore vesicles (LDCVs). The larger LDCVs typically have diameters around 150−300 nm and an inner ...

We report the lithographic microfabrication of a movable thin film microelectrode array (MEA) probe consisting of 16 platinum band electrodes placed on top of a supporting borosilicate glass substrate. These 1.2 μm wide electrodes were tightly packed and positioned parallel in two opposite rows within a 20 μm × 25 μm square area and with a distance less than 10 μm from the edge of the glass substrate. We demonstrate the ability to control and place the probe in close proximity to the surface of adherent bovine chromaffin cells and to amperometrically record single exocytosis release events with high spatiotemporal resolution. The two-dimensional position of single exocytotic events occurring in the center gap area separating the two rows of MEA band electrodes and that were codetected by electrodes in both rows was determined by analysis of the fractional detection of catecholamine released between electrodes and exploiting random walk simulations. Hence, two-dimensional electrochemical imaging recording of exocytosis release between the electrodes within this area was achieved. Similarly, by modeling the current spikes codetected by parallel adjacent band electrodes positioned in the same electrode row, a one-dimensional imaging of exocytosis with submicrometer resolution was accomplished within the area. The one- and two-dimensional electrochemical imaging using the MEA probe allowed for high spatial resolution of exocytosis activity and revealed heterogeneous release of catecholamine at the chromaffin cell surface.