Mechanosensing is a ubiquitous process to translate external mechanical stimuli
into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play
an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are
mainly examined by invasive or indirect techniques, such as electrophysiological analyses and
cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent
reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that
GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell
level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the
plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of
beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of
Piezo1 activity in mechanochemical feedback loops during development, homeostatic
regulation, and disease.