Multiple rounds of structure-guided design have made GCaMPs the most widely used protein calcium sensors11,16,17. But past efforts in optimizing GCaMPs and other indicators of neuronal function were limited by the throughput of quantitative and physiologically relevant assays. Because neurons have unusually fast calcium dynamics and low peak calcium accumulations4, sensors designed to probe neuronal function are best tested in neurons11,13,23,24, rather than in non-neuronal systems, most of which show much slower and larger calcium changes19. We thus screened GCaMP variants produced by mutagenesis in neurons, and subsequently validated lead sensors in several in vivo systems.
