Microscopy of Bioelectric Potentials using Electrochromism

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.

💡 Research Summary

The authors present a novel implementation of electrochromic optical recording (ECORE) that integrates a high‑numerical‑aperture (NA 1.49) microscope objective to achieve label‑free, non‑invasive measurement of cellular bioelectric activity with single‑cell spatial resolution. Traditional ECORE setups rely on a prism to couple light into a PEDOT:PSS‑coated glass slide, a geometry that is difficult to align and requires separate imaging optics. By using a single high‑NA objective both to deliver the illumination at super‑critical angles and to collect the reflected light, the authors simplify the optical train and enable simultaneous imaging of the specimen.

A super‑luminescent diode (SLD) centered at 510 nm serves as the illumination source. Its short coherence length (~8 µm) suppresses interference from parasitic reflections within the multi‑element objective, a major source of technical noise in previous designs. The beam is split by a Wollaston polarizer; one arm provides a reference to a differential photodetector while the other is expanded and directed through the objective onto the PEDOT film. The angle of incidence at the sample plane is tuned by laterally translating a 500 mm focal‑length lens that focuses the beam onto the back focal plane of the objective, allowing straightforward adjustment of the super‑critical illumination angle.

PEDOT:PSS films of varying thickness (25–94 nm) are electro‑deposited on indium‑tin‑oxide (ITO) coated glass coverslips. Optical characterization using a 1‑mV, 1‑Hz square wave applied across the film shows that the normalized reflectance change (ΔR/R) increases with thickness, while the absolute reflectance decreases. An optimal thickness of ~50 nm balances a strong electrochromic response with sufficient reflected power, avoiding excessive heating of the sample.

Noise performance is evaluated by recording a 100‑second trace of the 1‑mV square wave. The voltage spectral density reveals that low‑frequency noise is about an order of magnitude above the shot‑noise limit, indicating room for improvement, yet the overall signal‑to‑noise ratio (SNR) for each cycle averages 334. From this, the detection limit (unit SNR) is estimated at 3 µV, marginally better than the previous best ECORE record (≈3.3 µV).

The system is applied to human induced pluripotent stem cell‑derived cardiomyocytes. The recorded extracellular action potentials display the expected periodicity of spontaneous beating, with a sharp initial spike followed by a broader, longer‑duration component. The illumination spot on the sample, defined by the objective’s point spread function, measures 12.9 µm × 9.4 µm (1/e² radii), enabling interrogation of sub‑cellular regions. The authors note that further reduction of the spot size to ~3.9 µm (simple illumination) or ~138 nm (annular illumination) is possible, though higher irradiance may perturb or damage cells, limiting recording duration.

Overall, the microscope‑based ECORE platform delivers a detection limit of 3 µV, single‑cell spatial resolution, and the ability to acquire conventional bright‑field images simultaneously. By eliminating the need for fluorescent voltage indicators, it avoids photobleaching, phototoxicity, and potential alterations of membrane capacitance. The approach is readily adaptable to existing high‑NA microscopy facilities, opening the door for broader adoption in electrophysiology, drug screening, and tissue‑engineered construct monitoring. The work demonstrates that integrating electrochromic sensing with modern microscopy can provide a powerful, label‑free tool for real‑time bioelectric studies.