A near-infrared genetically encoded calcium indicator for in vivo imaging

While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared fluorescent proteins, we engineered a near-infrared FRET-based genetically encoded calcium indicator (iGECI). iGECI exhibits high brightness, high photostability, and up to 600% increase in fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity, and electrically and optogenetically induced firing. We validated iGECI performance up to a depth of almost 400 μm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with ~3 μm lateral and ~25–50 μm axial resolution. Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.