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

Nature Biotechnology volume 39, pages 368–377 (2021)Cite this article

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Abstract

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.

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Fig. 1: Characterization of iGECI in vitro and in HeLa cells.
Fig. 2: Characterization of iGECI in dissociated mouse neurons.
Fig. 3: Oblique light-sheet functional imaging of iGECI in acute brain slices.
Fig. 4: Spectral multiplexing of iGECI with GCaMP6s or the ChR2 optogenetic actuator in acute brain slices.
Fig. 5: In vivo imaging of iGECI using hybrid photoacoustic and fluorescence microscopy.
Fig. 6: iGECI reports visually evoked and spontaneous neuronal activity in vivo.

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Data availability

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.

Code availability

Acquisition and analysis code will be available on GitHub or on reasonable request.

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Acknowledgements

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.

Author information

Author notes

  1. Anton A. Shemetov

    Present address: Autonomous Therapeutics, Inc., New York, NY, USA

  2. Liming Nie

    Present address: Department of Radiology and Optical Imaging Laboratory, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China

  3. These authors contributed equally: Anton A. Shemetov, Mikhail V. Monakhov.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA

    Anton A. Shemetov, Mikhail V. Monakhov, Mikhail E. Matlashov, Daria M. Shcherbakova & Vladislav V. Verkhusha

  2. Department of Physics, University of California, Berkeley, Berkeley, CA, USA

    Qinrong Zhang & Na Ji

  3. Department of Neurobiology, Weinberg School of Arts and Sciences, Northwestern University, Evanston, IL, USA

    Jose Ernesto Canton-Josh, Manish Kumar & Yevgenia Kozorovitskiy

  4. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Maomao Chen & Junjie Yao

  5. Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, China

    Maomao Chen & Liming Nie

  6. Department of Anesthesiology, Duke University, Durham, NC, USA

    Xuan Li & Wei Yang

  7. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Yevgenia Kozorovitskiy

  8. Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Vladislav V. Verkhusha

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Contributions

V.V.V., D.M.S. and A.A.S. conceived the project. A.A.S. developed iGECI, and with M.E.M., performed in vitro characterization. M.V.M. characterized iGECI in dissociated neurons. J.E.C.-J., M.K. and Y.K. performed experiments in brain slices using a custom-designed and custom-built SOPi microscope. M.C., L.N. and J.Y. constructed and performed the hybrid photoacoustic and fluorescence microscopy experiments. X.L. and W.Y. developed the transgenic Emx1–hM3Dq mouse model. Q.Z. and N.J. characterized iGECI in vivo with two-photon microscopy. V.V.V., A.A.S., D.M.S., J.Y., Y.K. and N.J. designed the experiments, analyzed the data and wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Vladislav V. Verkhusha.

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Supplementary Table 1 and Supplementary Figs. 1–15

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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 39, 368–377 (2021). https://doi.org/10.1038/s41587-020-0710-1

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