<p>Studying ion channel activity and signalling interactions within cells are key tasks in neuroscience. Electrophysiological activities are typically measured with patch-clamp or voltage-sensitive imaging. Unfortunately, these techniques suffer from a trade-off between single-channel sensitivity and high-throughput detection. Here we introduce a label-free electrochemically modulated interferometric scattering microscope (EM-iSCAT) to measure ion channel activity on live cells at both the whole-cell and single-channel levels. We visualize the cellular responses dynamics to osmotic stimulation and record open–close trajectories of single receptor channels with a frame rate of 1.5 kHz. We also localize and distinguish different types of ion channels, including Na⁺, K⁺ and Ca²⁺, on the cell membrane and monitor spatio-temporal heterogeneous responses between different cells in a network. The high-throughput and single-channel sensitivity of EM-iSCAT microscopy enables the simultaneous monitoring of the activity of individual channels, their localization and clustering in the cellular community. EM-iSCAT has the potential to enable the study of any type of ion channel and, more broadly, cell communication pathways mediated by ion channels.</p>