Sub-second extracellular impedance measurement of epithelial cell monolayers using step excitations and time-domain analysis

Electrochemical impedance spectroscopy (EIS) is a versatile technique in epithelial biology, offering quantitative insights into barrier integrity, morphology, and apical-basolateral polarity non-invasively through measurements of transepithelial resistance (TER/TEER), capacitance (TEC), and membrane ratio (α). However, due to broad-spectrum frequency sweeps, EIS typically demands tens of seconds per measurement, limiting its applicability to faster biological phenomena. We present Time-domain Epithelial Impedance Measurement (TEIM), a method enabling sub-second extracellular impedance measurements of epithelial monolayers by combining step current excitations and time-domain voltage transient analysis while bypassing Fourier transforms. We experimentally demonstrate TEIM's ability to measure TER/TEER, TEC, α, and model-derived impedance spectrum every ∼0.3 s, which represents ∼100-fold time resolution improvement over EIS. TEIM's accuracy and precision were benchmarked against EIS using electrical circuits and human bronchial (16HBE) and colorectal (Caco-2) epithelial cell lines, yielding average errors for TER, TEC, and α ranging from 0.17-3.55%, 1.13-8.96%, and 0.59-26.35%, respectively. Applying TEIM to monitor Caco-2 responses to saponin revealed, for the first time, smoothly gated double-exponential transient TER and TEC dynamics too rapid to be adequately captured by EIS. Overall, TEIM offers a capable framework for probing rapid cellular electrophysiology, and offers a high-resolution alternative for dynamic assays in biological and pharmacological research.