In constant-potential amperometry, dopamine is rapidly and quantitatively oxidized after it is released under an electrode and the current is measured as a function of time. The resultant current vs. time trace is characterized for each release event.11,12
Amperometric detection yields both high temporal (~100 µs) and spatial resolution (~5 µm) due to the small physical size of the electrochemical probe. In our amperometric imaging experiments, the current-time response at each individual microelectrode is equivalent to those obtained at standard single carbon-fiber based electrochemical detection. shows a typical 15-s current-time response at a single PC12 cell. The current transients are detected after cells are stimulated using high K+
(100 mM) solution. shows spatial heterogeneity in single-cell exocytosis. For example, electrode E in records more numbers of events than other electrodes in the same array covering different parts of the cell.
Figure 1 (a) A 15-s amperometric imaging response of a single PC12 cell after a 5-s K+ stimulation, and (b), a 15-s voltammetric imaging response on a single PC12 cell using a carbon-fiber MEA. Electrodes in the array are labeled A-G with G being the middle electrode. (more ...)
Fast-Scan Cyclic Voltammetry Imaging
FSCV has been extensively used to measure neurochemicals released at single cells and in the central nervous system with carbon-fiber microelectrodes. FSCV-based methods add significant chemical resolution and sub-second temporal resolution to these measurements. In our experiments, FSCV has been carried out on individual PC12 cells using the seven individual carbon-fiber microelectrodes in the same array style. The advantage of utilizing FSCV with MEAs on a single cell is that it allows acquisition of chemical information in addition to high spatial resolution in electrochemical imaging.
is a 15-s time duration of a voltammetric imaging response (i.e. FSCV color plots) on a single PC12 cell using a carbon-fiber MEA. Each color plot represents a FSCV response from a 5-Sm-diameter area from the same PC12 cell. The current-time responses at the oxidation-peak potential (~0.7 V vs Ag/AgCl, at our scan rate, ν = 1500 V/s) for each color plot are given in Figure SI1a
. A comparison of a representative cyclic voltammogram (Figure SI1b
) and a voltammogram obtained from a dopamine solution at the same experimental condition indicate that the secreted molecule is dopamine. also shows that all exocytotic events show the same oxidation and reduction potentials, indicating that dopamine molecules have been released during this recording period.
Importantly, a comparison of the FSCV responses from all seven microelectrodes also shows that some exocytotic events occur at the same time at different electrode locations on the cell. One possibility is that multiple exocytotic events are being co-detected at different electrodes; however, it is possible that separate events cannot be temporally separated from each other due to their relatively long time duration. For example, an exocytotic signal is observed in at channel B at about 4.5 s. Simultaneous signals are also detected on all the other channels except channel A. Similarly, a transient is observed from channel A at about 8 s, with signals simultaneously detected at channels E, F, and G. Overlapping signals are also observed at several other time spots.
display a comparison of the temporal resolution of amperometry and FSCV in MEA-based electrochemical imaging on single PC12 cells. The results for amperometry and FSCV imaging reveal several distinct differences in the MEA experiments. The temporal resolution in amperometric imaging is again significantly higher than in FSCV. The peak width in amperometry is about 5 ms for PC12 cells. However, FSCV signals under the conditions used here have typical half widths of about 200–300 ms, which are more than 10 times longer than in amperometry. As a result, there is a greater possibility in FSCV for concurrent or overlapping signals to occur at an individual electrode as well as crosstalk between electrodes (unoxidized dopamine molecules have a longer time to diffuse to other microelectrodes). Thus, there are more simultaneous signals observed in voltammetric imaging experiments than in amperometry. In amperometric imaging, it is very unusual to observe simultaneous exocytotic signals from three or more locations on the same cell. However, exocytotic events are often detected from more than three locations in FSCV-type imaging experiments.
Figure 2 A comparison of the temporal resolution of FSCV and amperometry: (a) a 4-s amperometric response of a single carbon-fiber microelectrode on a PC12 cell, and (b), a 4-s trace of an FSCV recording on a single PC12 cell showing the peak current as a function (more ...)
Electrochemical signals are generated differently in amperometry and FSCV for obvious reasons. As illustrated in , in amperometry, electroactive neurotransmitters are quantitatively oxidized by the microelectrochemical probe placed in close proximity to the cell. Thus, the effective surface concentration of redox neurotransmitters approaches zero at the electrode surface under amperometric conditions. This generates a large concentration gradient between the site of release and the electrode surface (about 0.1 M inside the vesicle to essentially 0 at the electrode over a distance of ~200 nm13,14
) resulting in a large flux of neurotransmitters perpendicular to the cell surface. The diffusion of neurotransmitters in parallel to the cell membrane is slow compared to the oxidative consumption. As neurotransmitters released from underneath the electroactive surface of a specific electrode are oxidized quickly, this results in no significant crosstalk at adjacent electrodes. Our numerical simulation results show that even when neurotransmitters are released from in between two adjacent electrodes, they are mainly detected on the electrode closer to the fusion pore.
Figure 3 A schematic drawing of the concentration of released dopamine from a single vesicle in two different electrochemical recordings: FSCV and amperometry. The diagram shows the change in concentration of dopamine at early (µs to ms) and later (ms (more ...)
In FSCV experiments, however, electrochemical detection is obviously based on cycling oxidation and reduction of electroactive species, thus depletion does not occur as rapidly as for amperometry, but is dependent on diffusion out from under each electrode and degradation via coupled chemical reactions. shows released dopamine molecules being detected by repeated oxidation/reduction by FSCV at the individual electrode surfaces in the MEA. Here we assume any coupled chemical reactions are negligible so neurotransmitters are not consumed during electrochemical detection. This diffusion of neurotransmitters parallel to the cell membrane and electrode surface is generally slow compared to the time scale of the exocytosis event leading to significant broadening of FSCV signals. Additional diffusion resistance might result from adsorption of dopamine on carbon surfaces.15
In addition to broadening the response under an individual electrode, the lack of electrochemical consumption of the released transmitter in FSCV experiments means that molecules released from under one electrode can diffuse to the adjacent electrodes leading to significant crosstalk. This is an important issue in spatial resolution when using arrays of electrodes.
In order to better understand the differences in the temporal resolution and potential crosstalk in both amperometric and voltammetric imaging experiments, we simulated the electrochemical current at microelectrode arrays resulting from molecular diffusion from release via an open fusion pore (intermediate of exocytosis). The simulation is based on three-dimensional diffusion in a defined geometry involving both the cell and a microelectrode array placed above the cell membrane.
A cross-section of the geometry of the electrochemical cell used for the simulation is shown in (not drawn to scale). For the simulation, a PC12 cell is represented by the black area measured as 20-µm in diameter and 10-µm in height. A cylindrical fusion pore is formed between an intracellular vesicle (150-nm-diameter10
) and the cell membrane (5-nm thick). The position of the release site or fusion pore is measured about L
nm away from the center of the PC12 cell, which is varied to study the crosstalk as a function of the position of the release site. A microelectrode array is positioned above the cell membrane with a fixed cell/electrode gap measured of about 200 nm.13,14
The microelectrode-array contains seven microelectrodes, with each microelectrode having 5-µm tip diameter. The distance between adjacent electrodes (edge to edge) is assumed to be 1 µm. The total diameter of the array and the height of the microelectrode-array have been set to be 20 µm. The dimension of the electrochemical cell is much larger than both the size of the fusion pore through which release occurs and the electrode/cell gap, thus semi-infinite diffusion prevails outside the gap. Here, the cell and the electrode are placed in a solution, which measures 100-µm in diameter and 60-µm in height. The concentrations of dopamine inside and outside the vesicle have been set to be 150 mM and 0 mM, respectively, at time = 0 of the simulation. The intravesicular concentration prior to release was then calculated based on the number of dopamine molecules detected after release in amperometry and the volume of the vesicle in the simulation.
Figure 4 (a) The geometry of the electrochemical cell utilized for numerical simulations (cylindrical model through the electrode array with the cell below); (b) and (c), a comparison of the simulated amperometric response vs. a typical amperometric response. (more ...)
Exocytosis is a dynamic and complex process involving Ca2+
induced protein-protein interaction, and the opening and the enlargement of the fusion pore.16
Neurotransmitters are released after the opening of the fusion pore, quickly reaching a maximum of diffusive flux followed by a decay of the flux to zero.17
We were unable to model the entire dynamic change in fusion pore opening. However, we have successfully modeled both the decay of the amperometric current, which is the major part of the entire amperometric signal, and the shape of the FSCV signal using a fixed geometry of the fusion pore. We thus believe we can utilize the same simulation to evaluate the temporal resolution and potential crosstalk in the electrochemical imaging experiments. Our simulation results from both the amperometric signal and the FSCV signal give good agreement with the experimentally recorded signals.
Numerical Simulation of the Amperometric FSCV Signals
Although it is not the point of this paper, an important consideration in these simulations is the size of the fusion pore (the opening between the vesicle and the cell membrane during fusion). This was obtained by simulating a typical amperometric current-time response. We simulated the change in the flux of dopamine at the electrode surface as a function of time and then converted it into amperometric current using a 150 mM dopamine concentration. The diameter of the fusion pore was varied to make a comparison to a typical amperometric signal. displays different amperometric responses relative to the simulation when the diameter of the fusion pore was varied between 2 and 8 nm. The decaying part of the signal in the simulation was compared to the recorded signals as shown in . The simulated peaks shown in demonstrate that the amperometric current is dependent on the size of the fusion pore. The magnitude of the amperometric peak is larger when the fusion pore is larger corresponding to a larger diffusive flux. is typical signal recorded from amperometric recordings with the MEA at single PC12 cells (additional traces are provided in the supporting information, Figure SI2
). When comparing the simulations in with the experiment, we found that most of the typical signals have peak shapes that fall between those simulated with the diameter of the fusion pore is between 4 and 6 nm. Hence, we used 6 nm for the subsequent simulations. This value is consistent with the work of Amatore and coworkers showing that the pore opening angle appears to be too small to represent full opening.18
The same geometric parameters and similar dopamine concentrations in and outside the fusion pore were used for the simulations of the FSCV peak for dopamine and the simulations of amperometry. The only difference was in the boundary conditions used at the microelectrodes. In the FSCV experiments, the dopamine molecules are not consumed. Thus, the boundary condition at the microelectrode surface was set to be a constant flux = 0 and the simulation of the voltammetric signals has been carried out using a thin-layer-cell type model.19
This approach is valid as the gap between the electrode and the cell membrane is much smaller than the diffusion distance during the time course of the voltammetric experiment.19
In the simulation, the dopamine molecules were allowed to diffuse freely after they are secreted from the fusion pore. The number of the dopamine molecules underneath each microelectrode can be integrated from the simulation at any given time whereby the FSCV peak current was calculated by using the number of the molecules present under each electrode,19
is the peak current in FSCV, n
is the number of electrons transferred per dopamine molecule, F
is the Faraday constant, R
is the gas constant, T
is the temperature, N
is the number of dopamine molecules present underneath a single carbon fiber microdisk and ν is the sweep rate. is a simulated FSCV signal when the exocytotic release is located at the center of the electrode. shows an example of two typical FSCV signals with current at the voltammetric peak plotted vs. time. This approach must be qualified. We chose “typical” events here as average events will include those at different points from the electrode center. One can immediately notice that the peak current and the overall shape of the simulated response are in good agreement with the experimental recordings except that the simulated data show transients that are much faster than observed in the experimental recordings. In fact, the peak width in the simulation is about 1/3 than that in the experiment (predicted diffusional transport is approximately 9 times faster than observed). There are two possible reasons for this observation, which might contribute to the faster diffusion of dopamine in the simulation. First, the roughness of the cell membrane and the electrode surface might increase the diffusion length, or decrease the apparent diffusion coefficient.20
Second, it has been reported that dopamine strongly adsorbs at carbon electrodes,15
which might effectively slow down the apparent diffusion of the molecules.
The comparison between the recorded electrochemical data and the simulation reveals that the temporal resolution in electrochemical imaging can be qualitatively understood by release dynamics coupled to the diffusion of dopamine molecules in the electrode/cell gap. In amperometry, where dopamine is rapidly and completely oxidizedto the orthoquinione, the temporal resolution is only limited by how fast dopamine molecules are released from the fusion pore. In FSCV, the temporal resolution mainly depends on the diffusion of the dopamine molecules away from the electrode/cell gap. When this diffusion is to the space under an adjacent electrode, then electrochemical crosstalk occurs.
To analyze the potential for crosstalk in amperometric imaging, the position of the release site (the star in the insets of ) was varied between the microelectrode in the center (black circle) and two adjacent electrodes (Red circle and blue circle), as shown in . Figure parts 5a through 5c show the amperometric response at three adjacent electrodes as a function of the position of the release site. shows the responses at each microelectrode when the release site is present at the mid-point of the center electrode. One can see that there is no response at any other electrodes in the same array when release occurs at the center of a given microelectrode in the array. shows the response of three electrodes when release occurs under the center electrode, but ~50-nm away from the edge. The response at the adjacent electrode closest to the location of release shows about 2 percent the peak current observed under the center electrode and no current is observed at the other adjacent electrode on this current scale. Thus there is very little crosstalk in amperometric imaging when the release site is located underneath one microelectrode in the same array.
Figure 5 Simulated amperometric response at three adjacent microelectrodes in the MEA when the site of exocytotic release is localized at three different locations under the center carbon surface. The “*” in each figure indicates the location of (more ...)
When release occurs under the insulating material in between two microelectrodes, the simulation shows that the current at each electrode is dependent on their distances to the release site. displays the simulated amperometric response at three electrodes when the release site is present in between the center electrode (black) and the edge (blue) one. The currents at the center electrode (black) and the edge electrode are comparable when release occurs in the middle of the two microelectrodes. The current at the other edge electrode (red) is negligible as compared to the other two. Another important fact is that the two signals are comparable only when the release location is very close to the middle of the two microelectrodes. Therefore, crosstalk only occurs when the release occurs right at the middle of two adjacent electrodes.
When the location of release is close to the center of three adjacent microelectrodes, the simulated signals are comparable but are much smaller than the signals recorded when the vesicle is underneath the electrodes. In fact, the average peak current in this case is only ~10% of the current when the vesicle is present under a single microelectrode.
The simulations suggest that strong crosstalk is almost always present in FSCV imaging no matter where the location of release is found. shows the simulated FSCV peak current as a function of time when the release site is present at the center of the center electrode (black). It is immediately apparent that the signal at the adjacent electrode is 20–30% of the center electrode, indicating strong crosstalk. When the release site is not present under an electrode, the crosstalk is even stronger. shows the simulated signals when the release site is in between two adjacent electrodes. The signals are approximately the same for the two adjacent electrodes, which is about 25% of the signal when the vesicle is right under the microelectrode. Thus under the current conditions, the crosstalk can always be observed in FSCV type electrochemical imaging. This is in fact not surprising and has been reflected in our single-cell recording using FSCV, but we use the simulation to quantify and verify this effect here. Varying the sweep range of the applied potential could possibly eliminate the crosstalk and increase the temporal resolution in FSCV type experiments.
Figure 6 Simulated FSCV response at two adjacent electrodes when (a) the location of release is in the center of one electrode, and (b) when the location of release is between the two electrodes. The “*” in each figure indicates the location of (more ...)