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 (NIR) fluorescent proteins (FPs), we engineered an NIR Forster resonance energy transfer (FRET)-based genetically encoded calcium indicator (iGECI). iGECI exhibits high levels of brightness and photostability and an increase up to 600% in the fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity and electrically and optogenetically induced firing. We validated the performance of iGECI up to a depth of almost 400 mu m 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 resolutions of similar to 3 mu m (lateral) and similar to 25-50 mu m (axial). 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.

Automated summary

The newly designed near-infrared genetically encoded calcium indicator performs well in neuroscience, allowing simultaneous detection of neuronal and hemodynamic activities with high brightness, photostability, and increased fluorescence response rate. It enables deep-tissue imaging, is suitable for dissociated neurons and in vivo mouse experiments, and allows for detection of spontaneous and evoked neuronal activity in the mouse visual cortex with up to 25% fluorescence changes.