Far-red chemigenetic kinase biosensors enable multiplexed and super-resolved imaging of signaling networks.

Fluorescent biosensors have advanced biomedical research by enabling direct live-cell measurements of signaling activities. However, current technology offers limited resolution and dimensionality, impeding our ability to resolve and interrogate spatiotemporally regulated networks of signaling activities. Here we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling tag, HaloTag7, with far-red-emitting synthetic fluorophores. This technology enables both four-dimensional activity imaging and functional super-resolution imaging using stimulated emission depletion and other high-resolution microscopy techniques, permitting signaling activity to be detected across scales with the necessary resolution. Stimulated emission depletion imaging enabled the investigation of protein kinase A activity at individual clathrin-coated pits. We further demonstrate imaging of up to five analytes in single living cells, an increase in the dimensionality of biosensor multiplexing. Multiplexed imaging of cellular responses to the activation of different G-protein-coupled receptors (GPCRs) allowed quantitative measurements of spatiotemporal network states downstream of individual GPCR-ligand pairs.