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 Förster 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 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 resolutions of ~3 μm (lateral) and ~25–50 μm (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.
The main data supporting the findings of this study are available within the article and its Supplementary Information. Additional data are available from the corresponding author on reasonable request. GenBank accession numbers are MT997078 and MT997079 for the iGECI and iGECI-NES (nuclear exclusion sequence) constructs, respectively. Plasmids encoding these constructs will be available on Addgene.
Acquisition and analysis code will be available on GitHub or on reasonable request.
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We thank O. Oliinyk (University of Helsinki, Finland) and A. Kaberniuk (Albert Einstein College of Medicine) for useful suggestions, G. Robertson (Keyence Corporation of America) for technical support and the Biological Imaging Facility of Northwestern University for access to the confocal microscope. This work was supported by grants GM122567, NS103573, NS115581 (all to V.V.V.), EY030705 (to D.M.S.), EB028143, NS111039, EB027304, CA243822 (all to J.Y.) and MH117111 and NS107539 (both to Y.K.) from the National Institutes of Health; 18CSA34080277 from the American Heart Association (to J.Y.); a Beckman Young Investigator Award, a Searle Scholar Award and a Rita Allen Foundation Award (all to Y.K). J.E.C.-J. is a T32 NS041234 fellow.
The authors declare no competing interests.
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Shemetov, A.A., Monakhov, M.V., Zhang, Q. et al. A near-infrared genetically encoded calcium indicator for in vivo imaging.
Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0710-1