Chromaffin cells from the adrenal gland are a widely used model system to study neuronal exocytosis, and the fusion pore properties in these cells have been studied in great detail (3-6). Release of single vesicles (quanta) of epinephrine or norepinephrine can be detected electrochemically with a carbon fiber electrode (CFE) as amperometric spikes (7,8). These spikes are often preceded by a so-called foot signal (8), which is due to slow discharge of catecholamines through a narrow fusion pore (3) and indicates the time of fusion pore formation and expansion.To obtain not only temporal but also spatial resolution of opening and expansion of single fusion pores in chromaffin cells, we fabricated electrochemical detector (ECD) arrays of four Pt electrodes patterned on glass coverslips (9). For quantal events, the oxidation currents recorded from the four electrodes allow the desired determination of fusion pore dynamics with millisecond time resolution as well as localization of the fusion event.Simultaneous observation of the cell by fluorescence microscopy can be performed. Here we show that the spatiotemporal assignments of single exocytotic events obtained by electrochemical imaging of fusion events correlate with release of a fluorescent vesicle marker during exocytosis in chromaffin cells. Materials and MethodsECD Array. Pt conductors insulated by photoresist were fabricated photolithographically on standard microscope coverslips No. 1.5 (160-190 m thick) as described in ref. 9, forming a fourelectrode ECD array. Each ECD array consists of a square window Ϸ10 ϫ 10 m 2 in size in the insulating 0.5-m-thick photoresist. The active electrodes are formed by the tips of the four Pt conductors, Ϸ3 m wide and 150 nm thick, that protrude into the corners of the square. The area and shape of the electrode arrays were imaged and quantified by using atomic force microscopy and optical microscopy.Cell Preparation and Recording Conditions. Bovine adrenal glands were obtained from a local slaughterhouse and prepared as described in ref. 10. Cells were cultured on 12-mm coverslips that were coated with 0.02% poly(D)-lysine and used on days 1-10. Before the experiment, a small coverslip with cells attached was placed in a 35-mm dish with 1 ml of bath solution containing 150 mM NaCl, 10 mM Hepes, 5 mM CaCl 2 , 5 mM KCl, 2 mM MgCl 2 (pH 7.25) (318 mosM) and supplemented with 3 M acridine orange (Molecular Probes) for 15 min in the dark. An ECD array coverslip was mounted on a custom-built stage on a Zeiss 135TV microscope, and the coverslip with the cells was placed at some distance from the ECD array. Bath solution (150-200 l) was then added to cover the cells and the active electrode array. By using a patch pipette, a cell was lifted off the coverslip and manipulated over the center of an ECD array. Some cells were stimulated by including ionomycin in the pipette or adding ionomycin to the bath. Experiments were performed at room temperature.Electrochemical Recording and Analysis. The ECD electrodes were connected via their...

Nanobiotechnology is a field that utilizes the techniques of nano- and microfabrication to study biosystems or to use biological material and principles to build new devices. As an example we discuss the development of a nanofabricated electrochemical detector array that reveals the spatio-temporal dynamics of exocytosis in single chromaffin cells. In a quantal release event a single vesicle fuses with the plasma membrane releasing its contents through the fusion pore. The time-resolved amperometric currents measured by the individual electrodes detecting different fractions of the released molecules allow determination of the time course as well as localization of quantal events. Such a device may be applicable to study the correlation of exocytotic events with signalling events that could be simultaneously monitored by fluorescence microscopy.

Objective This study compared the effects of calcium chloride dihydrate (CaCl 2 .2H 2 O) on the physical properties and push-out bond strength of white Mineral Trioxide Aggregate (WMTA) and an experimental Malaysian Portland cement mixed with nano-zirconium oxide (nano-ZrO) [(radiopaque Malaysian Portland cement (RMPC). Mineral Trioxide Aggregate (MTA) was the first calcium silicate cement (CSC) introduced in dentistry, but up to date, it is an expensive cement with long setting time and causes tooth discolouration. Although Portland cement has been introduced as a potential substitute to MTA, it still faces some challenges such as long setting time and lack of sufficient radiopacity. Methods Four groups [WMTA, RMPC, fast-set WMTA (FS-WMTA) and fast-set RMPC (FS-RMPC)] were prepared. Initial setting time was evaluated using Vicat apparatus. The pH was measured at seven-day intervals. For discolouration potential, cements were packed in the pulp chamber of 46 extracted maxillary incisors. Spectrophotometric readings were obtained at seven-day intervals, and the rate of colour change (∆E) was recorded. For the push-out bond strength testing, cements were applied in 48 sectioned root samples, and the test was performed using universal testing machine at crosshead speed of 0.5 mm/min until bond failure. Statistical analysis was done according to the nature of each group of data using SPSS 26. Results Addition of CaCl 2 .2H 2 O decreased the initial setting times of both RMPC and WMTA significantly (p<0.05). The pH values of FS-WMTA and FS-RMPC were comparable to their non-accelerated counterparts ranging from 10 to 12. Discolouration effect was more obviously observed with WMTA and FS-WMTA with time compared to RMPC formulations. Push-out bond strength of the two materials also showed an increase with the addition of the accelerator, however, only FS-WMTA showed statistically significant difference compared to WMTA (p<0.05). Conclusion The addition of CaCl 2 .2H 2 O improves the physical and mechanical properties of the newly formulated RMPC and WMTA. The RMPC formulation overcomes the discolouration potential of WMTA.