Abstract
Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates that reduce fluorescence detection to near shot-noise levels. High-photon flux excitation can overcome photon-limited shot noise, but photobleaching and photodamage restrict the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, which is voltage imaging below the shot-noise limit. This framework involved developing positive-going voltage indicators with improved spike detection (SpikeyGi and SpikeyGi2); a two-photon microscope (‘SMURF’) for kilohertz frame rate imaging across a 0.4 mm × 0.4 mm field of view; and a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise-limited signals. Through these combined advances, we achieved simultaneous high-speed deep-tissue imaging of more than 100 densely labeled neurons over 1 hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.
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Data availability
The DNA sequences are available in GenBank under accession numbers OQ143892 (SpikeyGi) and OQ143893 (SpikeyGi2). The plasmid pAAV-hSyn-SpikeyGi2-kv2.1 (no. 82031) is available through Addgene. Experimental data are available at https://doi.org/10.12751/g-node.1b9v1w.
Code availability
Imaging analysis code is available at github.com/common-chenlab. DeepVID code is available at github.com/bu-cisl/DeepVID through GNU General Public License v3.0. Scope software code is available at rkscope.sourceforge.net through MIT license.
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Acknowledgements
We thank K. Khait for assistance in Scope software development; N. Manjrekar for assistance in photodamage experiments; Pieribone Laboratory scientific staff J. Wojciekofsky, L. Delgado, R. O’Brien and P. O’Brien for technical assistance; and the staff of the Pierce Laboratory Instrument shop, including J. Buckley, A. Wilkins, T. D’Alessandro, and A. DiRubba, for help with instrumentation. We thank Adam Cohen (Harvard University) for the kind gift of electrically active HEK293 cells. We thank Michael Z. Lin (Stanford University) for the generous gift of pcDNA3.1/Puro-CAG-ASAP3b and pcDNA3.1/Puro-CAG-ASAP3b-Kv2.1. This work was supported by grants from a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation (J.L.C.), the Richard and Susan Smith Family Foundation (J.L.C.), Elizabeth and Stuart Pratt Career Development Award (J.L.C.), the Whitehall Foundation (J.L.C.), NSF Neuronex Neurotechnology Hub NEMONIC no. 1707287 (J.L.C.), NIH New Innovator Award DP2NS111134 (J.L.C.), NIH BRAIN Initiative Awards R01NS109965 (J.L.C.), UF1NS107705 (J.L.C. and V.A.P.), U01NS128665 (J.L.C., L.T. and V.A.P.), R21EY030016 (L.T.), NIH R01NS126596 (L.T.), U01NS103517 (V.A.P), U01NS090565 (V.A.P), and DARPA N6600117C4012 (V.A.P.) and N660119C4020 (V.A.P.).
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Contributions
J.L.C. and V.A.P. initiated and supervised the study. J.P. engineered SpikeyGi and SpikeyGi2. J.P. carried out cell culture experiments and analyzed data. X.Y. designed, built, and characterized the SMURF two-photon microscope. C.L. developed DeepVID algorithm and analyzed performance. A.M.A. performed animal surgeries for slice and in vivo experiments. A.M.A. and I.G.D. performed slice experiments and analyzed the data. X.Y. and A.M.A. performed in vivo experiments. X.Y., A.M.A. and J.L.C. analyzed in vivo data. I.A.C., I.G.D., L.T., V.A.P. and J.L.C. provided input and guidance. J.P., X. Y., A.M.A., C.L., L.T., V.A.P. and J.L.C. prepared the manuscript.
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Nature Methods thanks Michael Häusser and the other anonymous reviewers for their contribution to the peer review of this work. Primary Handling editor: Nina Vogt, in collaboration with the Nature Methods team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Single-photon imaging of spontaneous activity in cultured mouse neurons expressing SpikeyGi or SpikeyGi2.
(a) Single-photon image of a SpikeyGi-expressing neuron (left). Fluorescence response to spontaneous neuronal activity imaged at 500 Hz (right). This experiment was performed 3 times with similar results. (b) Single-photon image of a SpikeyGi2-expressing neuron (left). Fluorescence response to spontaneous neuronal activity imaged at 500 Hz (right). This experiment was performed 5 times with similar results. Similar signals were recorded in three SpikeyGi and five SpikeyGi2 neurons. Scale bar: 5 µm.
Extended Data Fig. 2 SpikeyGi2 responses in vitro across neuronal cell types.
(a) Circuit diagram of recorded cell types in the anterior olfactory nucleus and main olfactory bulb. (b) Example of in vitro fluorescence in an anterior olfactory nucleus pyramidal neuron expressing SpikeyGi2 in response to 10 Hz and 50 Hz action potential trains. (c) Example of in vitro fluorescence in a main olfactory bulb periglomular neuron expressing SpikeyGi2 in response to10 Hz and 50 Hz action potential trains. Scale bar: 10 𝜇m. These experiments were performed 5 times with similar results.
Extended Data Fig. 3 Opto-mechanical design of SMURF 2P microscope.
(a, b) CAD design rendering of our fast microscope. (c) CAD design rendering of the beam splitter plate.
Extended Data Fig. 4 Example point spread function measurements for each beamlet in the SMURF 2P microscope.
Scale bar: 1 µm. Examples are shown from n = 7 beads, 1A; 10 beads, 2A; 10 beads, 3A; 10 beads, 4A; 11 beads, 1B; 9 beads, 2B; 10 beads, 3B; 10 beads, 4B.
Extended Data Fig. 5 Detected Crosstalk In Vivo.
Example of crosstalk from each beamlet observed across each detected subarea. In vivo images are of SpikeyGi expression at ~150 µm below the pial surface. Scale bar: 50 µm.
Extended Data Fig. 6 Analysis of DeepVID performance.
(a) Power spectrum analysis comparing DeepVID denoising against raw and 7-frame moving average for all frequencies (left) and 0–50 Hz (right). (b) Top panels show simulated motion traces in X- and Y-axis at three levels of residual motion artifacts corresponding to a maximum total displacement at (low) ±2, (medium) ±4, and (high) ±6 µm. (c) Power spectrum analysis of DeepVID denoising results at different motion artifact levels for all frequencies (left) and 0–50 Hz (right).
Extended Data Fig. 7 In vivo population imaging of SpikeyGi from SMURF 2P microscope.
(a) Fluorescence traces from simultaneous recordings across 129 S1 neurons from our fast microscope acquired at 803 Hz. Raw images were denoised with DeepVID. For visualization purposes, traces are detrended with 2.5 sec moving average and low pass filtered at 200 Hz. Red lines indicate air puff whisker stimulus. Detected action potentials are plotted in Fig. 5b. (b) Magnified view of example traces across different cells. Squares denote traces corresponding to shaded regions of the same color indicated in [a]. Red lines indicate air puff whisker stimulus.
Extended Data Fig. 8 In vivo patch recording in SpikeyGi2 expressing neuron.
(a) In vivo two-photon image of targeted S1 neuron (red) expressing SpikeyGi2 (green). Scale bar: 50 𝜇m. This experiment was performance once. (b) Fluorescence response to applied voltage steps. (c) Measured fluorescence response from example neuron overlaid with fluorescence responses measured in vitro (in vitro, n = 6 cells; 10 trials per step). Error bars; S.E.M.
Extended Data Fig. 9
Example SpikeyGi fluorescence traces from 5 neurons across 1 hour of in vivo imaging.
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Platisa, J., Ye, X., Ahrens, A.M. et al. High-speed low-light in vivo two-photon voltage imaging of large neuronal populations. Nat Methods 20, 1095–1103 (2023). https://doi.org/10.1038/s41592-023-01820-3
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DOI: https://doi.org/10.1038/s41592-023-01820-3
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