Brain heating induced by near-infrared lasers during multiphoton microscopy. A general approach to engineer positive-going eFRET voltage indicators. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. Kilohertz two-photon brain imaging in awake mice. Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics. Population imaging of neural activity in awake behaving mice. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo. Ultrafast two-photon imaging of a high-gain voltage indicator in awake behaving mice. Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Optical recording of action potentials and other discrete physiological events: a perspective from signal detection theory. Photon shot noise limits on optical detection of neuronal spikes and estimation of spike timing. Relationship between simultaneously recorded spiking activity and fluorescence signal in GCaMP6 transgenic mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations. 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 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. We investigated an alternative approach aimed at low two-photon flux, which is voltage imaging below the shot-noise limit. High-photon flux excitation can overcome photon-limited shot noise, but photobleaching and photodamage restrict the number and duration of simultaneously imaged neurons. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates that reduce fluorescence detection to near shot-noise levels. Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function.
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