Electrochemical imaging of fusion pore openings by electrochemical detector arrays

Abstract

Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of catecholamines near the cell surface and not due to slow dissociation from the granular matrix.

Fig. 1.

ECD array recording from a chromaffin cell. (A) Light microscope image of a chromaffin cell placed on top of an ECD array, with four electrodes labeled A through D. (B) Amperometric recordings from the four electrodes (channel A, red trace; channel B, green trace; channel C, black trace; channel D, blue trace) recorded from a single bovine chromaffin cell mechanically stimulated by a patch pipette in calcium containing external solution.

Fig. 2.

Analysis of an exocytotic event. (A) One exocytotic event from the experiment of Fig. 1 shown on an expanded time scale. Arrows mark start and end of the foot signal (black trace) indicating fusion pore opening (O) and expansion (E). (Inset) Foot signal on expanded current scale. (B) Running integrals of the amperometric currents provide the total charge detected by the individual electrodes. (C) Currents from the four electrodes after normalization of the amplitudes reveal differences in time course for the different signals. (D) Spatiotemporal correlation of secretion of catecholamines and release of the fluorescent vesicle marker acridine orange. (Left) Original sequence of fluorescence images. (Right) Difference images obtained by subtracting the average of 10 images preceding the sequence. The exposure times for each frame are indicated on the horizontal axes of A–C. The fluorescence “flash” in the difference images indicates the position of the exocytotic event. The release position calculated from the electrochemical signals (B) is indicated in D as a red cross. (Scale bar, 5 μm.)

Fig. 3.

Correlation between electrochemical and fluorescence imaging. (A) Outline of the detector map (red lines) used for the random walk simulations overlayed on image of the actual electrodes. The dashed blue circle indicates the central area with a 2-μm radius. (B) Difference images (a–v) of all 22 fluorescence flashes from this cell for which position assignments were obtained. The red crosses (at end of arrows) indicate the position assignments from electrochemical imaging. Difference image i shows the event of Fig. 2. In all cases the fluorescence flash appeared in the image frame immediately following the spike of catecholamine release measured electrochemically. (C) Comparison of the measured x coordinates obtained from electrochemical and fluorescence imaging. (D) Comparison of y coordinates. The dashed lines with slope 1 would be expected for perfect agreement between the two independent measurements. The red data points refer to events located outside the blue circle of Fig. 3A.

Fig. 4.

Integrated experimental (solid lines) and fitted (dashed lines) amperometric signals using the standard diffusion constant D = 6 × 10^–6 cm²/s (A) and a diffusion constant of D = 8 × 10^–7 cm²/s(B). (Insets) Time course of release resulting from the fits. All four traces were fitted simultaneously.