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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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.
Scanziani, M. & Hausser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009).
Leopold, A. V., Shcherbakova, D. M. & Verkhusha, V. V. Fluorescent biosensors for neurotransmission and neuromodulation: engineering and applications. Front. Cell Neurosci. 13, 474 (2019).
Ji, N., Milkie, D. E. & Betzig, E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat. Methods 7, 141–147 (2010).
Debarre, D. et al. Image-based adaptive optics for two-photon microscopy. Opt. Lett. 34, 2495–2497 (2009).
Rueckel, M., Mack-Bucher, J. A. & Denk, W. Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing. Proc. Natl Acad. Sci. USA 103, 17137–17142 (2006).
Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).
Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl Acad. Sci. USA 101, 10554–10559 (2004).
Palmer, A. E. et al. Ca2+ indicators based on computationally redesigned calmodulin–peptide pairs. Chem. Biol. 13, 521–530 (2006).
Thestrup, T. et al. Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat. Methods 11, 175–182 (2014).
Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).
Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).
Shcherbakova, D. M. et al. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nat. Commun. 7, 12405 (2016).
Yu, D. et al. A naturally monomeric infrared fluorescent protein for protein labeling in vivo. Nat. Methods 12, 763–765 (2015).
Matlashov, M. E. et al. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nat. Commun. 11, 239 (2020).
Qian, Y. et al. A genetically encoded near-infrared fluorescent calcium ion indicator. Nat. Methods 16, 171–174 (2019).
Shcherbakova, D. M., Cox Cammer, N., Huisman, T. M., Verkhusha, V. V. & Hodgson, L. Direct multiplex imaging and optogenetics of RhoGTPases enabled by near-infrared FRET. Nat. Chem. Biol. 14, 591–600 (2018).
Horikawa, K. et al. Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano. Nat. Methods 7, 729–732 (2010).
Bootman, M. D. & Berridge, M. J. Subcellular Ca2+ signals underlying waves and graded responses in HeLa cells. Curr. Biol. 6, 855–865 (1996).
Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).
Kumar, M., Kishore, S., Nasenbeny, J., McLean, D. L. & Kozorovitskiy, Y. Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging. Opt. Express 26, 13027–13041 (2018).
Kumar, M. & Kozorovitskiy, Y. Tilt-invariant scanned oblique plane illumination microscopy for large-scale volumetric imaging. Opt. Lett. 44, 1706–1709 (2019).
Kumar, M. & Kozorovitskiy, Y. Tilt (in)variant lateral scan in oblique plane microscopy: a geometrical optics approach. Biomed. Opt. Express 11, 3346–3359 (2020).
Herman, A. M., Huang, L., Murphey, D. K., Garcia, I. & Arenkiel, B. R. Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2. eLife 3, e01481 (2014).
Brunker, J., Yao, J., Laufer, J. & Bohndiek, S. E. Photoacoustic imaging using genetically encoded reporters: a review. J. Biomed. Opt. 22, 070901 (2017).
Yao, J. et al. Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe. Nat. Methods 13, 67–73 (2016).
Yao, J. et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action. Nat. Methods 12, 407–410 (2015).
Zhu, H. et al. Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice. Genesis 54, 439–446 (2016).
Manvich, D. F. et al. The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Sci. Rep. 8, 3840 (2018).
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).
Bouchard, M. B., Chen, B. R., Burgess, S. A. & Hillman, E. M. Ultra-fast multispectral optical imaging of cortical oxygenation, blood flow, and intracellular calcium dynamics. Opt. Express 17, 15670–15678 (2009).
Piatkevich, K. D. et al. Near-infrared fluorescent proteins engineered from bacterial phytochromes in neuroimaging. Biophys. J. 113, 2299–2309 (2017).
Girouard, H. & Iadecola, C. Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer disease. J. Appl. Physiol. 100, 328–335 (2006).
Fabiani, M. et al. Neurovascular coupling in normal aging: a combined optical, ERP and fMRI study. Neuroimage 85(Pt. 1), 592–607 (2014).
Liao, L. D. et al. Neurovascular coupling: in vivo optical techniques for functional brain imaging. Biomed. Eng. Online 12, 38 (2013).
Wang, L. V. & Yao, J. A practical guide to photoacoustic tomography in the life sciences. Nat. Methods 13, 627–638 (2016).
Gottschalk, S. et al. Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain. Nat. Biomed. Eng. 3, 392–401 (2019).
Piatkevich, K. D., Subach, F. V. & Verkhusha, V. V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nat. Commun. 4, 2153 (2013).
Challis, R. C. et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat. Protoc. 14, 379–414 (2019).
Beaudoin, G. M. 3rd et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741–1754 (2012).
Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).
Wardill, T. J. et al. A neuron-based screening platform for optimizing genetically-encoded calcium indicators. PLoS ONE 8, e77728 (2013).
Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).
Kozorovitskiy, Y., Peixoto, R., Wang, W., Saunders, A. & Sabatini, B. L. Neuromodulation of excitatory synaptogenesis in striatal development. eLife 4, e10111 (2015).
Xiao, L., Priest, M. F., Nasenbeny, J., Lu, T. & Kozorovitskiy, Y. Biased oxytocinergic modulation of midbrain dopamine systems. Neuron 95, 368–384 (2017).
Xiao, L., Priest, M. F. & Kozorovitskiy, Y. Oxytocin functions as a spatiotemporal filter for excitatory synaptic inputs to VTA dopamine neurons. eLife 7, e33892 (2018).
Edelstein, A. D. et al. Advanced methods of microscope control using µManager software. J. Biol. Methods 1, e10 (2014).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
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 39, 368–377 (2021). https://doi.org/10.1038/s41587-020-0710-1
Nature Methods (2021)
European Journal of Nuclear Medicine and Molecular Imaging (2021)