biostudies-literaturePlatisa JRichard and Susan Smith Family FoundationU.S. Department of Health & Human Services | NIH | National Eye InstituteNEI NIH HHSU.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and StrokeUnited States Department of Defense | Defense Advanced Research Projects AgencyWhitehall FoundationBrain and Behavior Research FoundationNINDS NIH HHSNational Science Foundation1095-1103https://www.ebi.ac.uk/biostudies/studies/S-EPMC10894646biostudies-literatureUnknown20(7)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 fNature methodsHigh-speed low-light in vivo two-photon voltage imaging of large neuronal populations.PMC10894646U01 NS090565U01 NS103517R01 NS083875U01NS103517R01NS1099651707287N660119C4020R21EY030016R01 NS109965R01 NS126596UF1NS107705R01 NS065110DP2NS111134UF1 NS107705DP2 NS111134U01NS090565R21 EY030016N6600117C4012U01NS128665U01 NS128665Ahrens AMChen IATian LDavison IGLiu CPieribone VAYe XPlatisa JChen JLfalseHigh-speed low-light in vivo two-photon voltage imaging of large neuronal populations.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 f2023-01-01T00:00:00Z2023 Jul2026-06-02T19:27:01.644Z2025-04-06T00:39:49.262ZS-EPMC108946463697354710.1038/s41592-023-01820-3