RT Journal Article SR Electronic T1 High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.12.07.471668 DO 10.1101/2021.12.07.471668 A1 Jelena Platisa A1 Xin Ye A1 Allison M. Ahrens A1 Chang Liu A1 Ichun Anderson Chen A1 Ian G. Davison A1 Lei Tian A1 Vincent A. Pieribone A1 Jerry L. Chen YR 2021 UL http://biorxiv.org/content/early/2021/12/09/2021.12.07.471668.abstract AB 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 which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4×0.4mm2 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 one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.Competing Interest StatementThe authors have declared no competing interest.