Sub-second Extracellular Impedance Measurement of Epithelial Cell Monolayers using Step Excitations and Time-domain Analysis

Extracellular electrochemical impedance spectroscopy (EIS) is emerging as a powerful technique in <i>in vitro</i> epithelial research, offering quantitative insights into barrier integrity, morphology, and apical-basolateral polarity noninvasively through metrics such as transepithelial electrical resistance (TER/TEER), transepithelial capacitance (TEC), and membrane ratio ( <i>α</i> ), respectively. However, due to the broad range of frequencies probed, EIS typically requires tens of seconds per measurement, limiting its ability to capture more rapid biological phenomena. We present Time-domain Epithelial Impedance Measurement (TEIM), a method for sub-second extracellular impedance measurements of epithelial cell monolayers based on step (Heaviside function) current excitation and time-domain analysis of the voltage transients, without the need for Fourier transforms. We experimentally demonstrate TEIM measuring TER, TEC, <i>α</i> , and model-derived impedance spectrum at ∼0.3 s sampling rate, which represents a 100- fold improvement in time resolution compared with traditional EIS. The accuracy and precision of TEIM were benchmarked against EIS on both electrical circuits and epithelial cell monolayers of immortalized Human Bronchial Epithelial (16HBE) and Human Colorectal Adenocarcinoma (Caco-2) (n = 3 for each), and average percent errors for TER, TEC, and <i>α</i> ranged from 0.17-3.55%, 1.13-8.96%, and 0.59-26.35%, respectively. Application of TEIM to monitor Caco-2 responses to saponin, a quick-acting pore-forming detergent, revealed smoothly gated double-exponential transient TER and TEC dynamics that were too rapid to be adequately captured previously using EIS. Overall, TEIM enables electrophysiology studies of rapid changes in epithelial cell culture models and possibly more complex <i>in vitro</i> models, holding promise for future applications in areas such as disease modeling, therapeutic development, and beyond.