Microscopy of Bioelectric Potentials using Electrochromism

TL;DR

This work tackles the challenge of noninvasively recording bioelectric potentials with high spatial resolution using a label-free optical method. It introduces microscope ECORE, integrating a high-NA objective and a PEDOT-based electrochromic interface to detect extracellular potentials while enabling concurrent imaging. The approach achieves a detection limit around 3 μV and demonstrates high-SNR recordings of extracellular cardiomyocyte action potentials, plus spatial mapping across subcellular regions of a single cell. The method simplifies the optical setup, enhances accessibility, and holds promise for real-time studies of excitable cells, drug effects, and stimulation paradigms.

Abstract

Studying the electrical signals generated by living cells is key to understanding numerous biological phenomena. Electrochromic optical recording (ECORE) uses the electrochromism exhibited by certain materials to noninvasively measure these signals in real time. In this work, we report on the development of ECORE based on a high-NA microscope objective. We demonstrate the recording of extracellular action potentials from cardiomyocytes with single-cell resolution and a high sensitivity of 3 μV, which compares favorably to the previous record for any ECORE setup. Combining ECORE with microscopy simplifies the optical setup, allows for the simultaneous imaging of specimens, and makes ECORE accessible to a broader community of researchers, allowing for a better understanding of the biological processes that are integral to life.

Figures (5)

• Figure 1: (a) Schematic drawing of microscope ECORE. M: mirror, $\mathrm{\lambda/2}$: half-wave plate. All components, except for the light source, are placed on an optical breadboard measuring $36 \times 24$ inches. (b) Detail showing the focused light at the sample. (c) A section of a microscope image (from a separate objective) showing a typical sample of cardiomyocytes. The dashed ellipses superposed on the image depict the $\mathrm{1/e^2}$ intensity radius spot sizes of various ECORE beams. From left to right: original ECORE PNAS, dual-color ECORE dualcolor, and microscope ECORE.
• Figure 2: (a) Detector output (in units of normalized change in the reflectance of the sample $\Delta R/R$) in response to a [number-unit-product=-]1, [number-unit-product=-]1 applied square wave at a bias voltage of 0mV (with respect to a standard Ag/AgCl electrode). A [number-unit-product=-]0.01 high-pass digital filter (first-order Bessel) has been applied to flatten the baseline. (b) $\Delta R/R$ in response to a [number-unit-product=-]1, [number-unit-product=-]1 (at zero bias) applied square wave as a function of the PEDOT film thickness (black circles and line). The reflectance $R$ of the samples is also shown (green triangles and line). (c) $\Delta R/R$ as a function of the amplitude of the applied square wave at zero bias. The line is a linear fit to the data described by the equation $y = [separate-uncertainty = true]{1.3696(23)e-3}{}\ x$. The inset shows data points near the origin.
• Figure 3: (a) Voltage spectral density (VSD) of a [number-unit-product=-]100-long microscope ECORE recording (black) in response to a [number-unit-product=-]1, [number-unit-product=-]1 square wave at zero bias. Peaks associated with the [number-unit-product=-]1 applied signal are visible and have been cropped to show the noise floor in more detail. The inset shows a zoomed-out version of the spectrum for lower frequencies. 600µW of light enters the microscope base and 44µW reaches the signal eye of the detector. The shot noise limit (see Appendix \ref{['appendix:shotnoise']}) corresponding to the physical filter is also shown (dashed red line). (b) The left panel shows a [number-unit-product=-]5 section from the signal that was used to generate the VSD in (a) after the application of [number-unit-product=-]0.01 high-pass (first-order) and [number-unit-product=-]280 low-pass (third-order) digital Bessel filters. A zoomed-in section is shown in the right panel and illustrates the procedure for determining the SNR of one cycle from the entire [number-unit-product=-]100 recording. The dashed black line represents the time value associated with the half-amplitude of the cycle and is determined by first applying a Gaussian filter to smooth the data and then finding the peak of its derivative. The red and green shaded sections show [number-unit-product=-]25 regions of the data 200ms on either side of the dashed line. Linear fits are performed in both of these regions. The average standard deviation of the two fits determines the noise associated with the cycle, while the amplitude difference between the average $\Delta R/R$ in each region determines the signal for this cycle. (c) The SNR results from fitting all cycles in the [number-unit-product=-]100 recording (left). The mean $\textrm{SNR} = 334.0$ is also shown (dashed red line). A histogram of these SNR measurements is shown on the right.
• Figure 4: (a) The ECORE signal from a cardiomyocyte is shown on the left. A zoomed-in section of the recording in shown on the right. The optical response from the electrical signal in the cells as well as the mechanical contraction are identified. (b) [number-unit-product=-]10 high-pass and [number-unit-product=-]280 low-pass filters (third-order Bessel) are applied to the data shown in (a). The right panel shows the effect of filtering: the electrical signal is still clearly visible but the mechanical contraction has been suppressed. (c) A 20µ solution of blebbistatin is added to a cardiomyocyte sample at $t = 0min$. The black trace shows the recorded signal, where a [number-unit-product=-]0.1 high-pass first-order Bessel filter has been applied to flatten the recording baseline. The red trace shows the signal after the application of the [number-unit-product=-]10 high-pass and [number-unit-product=-]280 low-pass filters described in (b). The black trace shows that the mechanical signal is reduced over time, while the red filtered trace confirms that the electrical signal persists.
• Figure 5: (a) A cardiomyocyte (center) and its neighboring cells in the field of view of the 1.49-NA objective. The dashed ellipses show the location of the probing beam. The beam was moved to a particular spot, where the ECORE signal was recorded for 60s before moving to another spot and repeating this process. (b) Sections of the recordings from the corresponding ellipses in (a) after the application of [number-unit-product=-]10 high-pass and [number-unit-product=-]280 low-pass filters (third-order Bessel). The recordings have been manually synchronized in time to facilitate comparison of the electrical signals in the different regions.