Low-Noise Fluorescent Infrared Detection of Single-Cell Cardiac Action Potentials

Abstract

Optical recordings with voltage-sensitive fluorescent dyes enable non-invasive action potential (AP) measurements, which are useful for high-throughput drug screening. However, optical measurements are hampered by dye rundown, photobleaching, and phototoxicity. Long-lasting infrared dyes overcome these limitations, but optical recordings from single-cells are difficult due to low signal-to-noise ratios (SNR). Using the voltage-sensitive infrared dye, Di-4-ANBDQBS (Cytovolt1), we developed an optical system consisting of matched hardware, biological, and dye components to produce a low-noise fluorescent infrared detection system for single-cell AP recording. We combined a high-sensitivity photodiode-based detector, a high-stability LED excitation source, and an optimized optical filter set for enhanced single-cell optical AP recordings. We validated our system by correlating membrane voltage to fluorescence under single-cell patch clamp, and showed the fluorescence signal is highly-linear with membrane voltage. We also optimized dye concentration and staining method along with emission/excitation wavelengths to yield maximum ΔF/F and thus maximum SNR. To correlate membrane voltages to optical voltage signals, we performed simultaneous patch clamp and optical recordings on HEK293 cells and human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs). Low-noise recordings, with minimal dye rundown, were obtained from single-cells. Optical signals were recorded in response to a voltage ramp, and averaged R2 values from linear fits of fluorescence versus voltage were 0.88+0.04, 0.97+0.007, 0.97+0.004 and 0.98+0.004 for 50, 75, 100 and 150 μM of Di‑4‑ANBDQBS, respectively, in HEK293 cells. ΔF/F measurements were 10.5+0.3%, 14.9+1.8%, 13.8+2.5% and 13.6+3.5% for 50, 75, 100 and 150 μM of dye, respectively. For hiPSC-CMs, ΔF/F measurements from simultaneous voltage and fluorescence of APs were up to 23.1% per 100 mV. Taken together, these data demonstrate the feasibility of high fidelity single cell voltage measurements in a simple system not requiring photomultiplier tubes or laser excitation.