Department of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA.
The John B. Pierce Laboratory, New Haven, CT, USA.
Nat Methods. 2023 Jul;20(7):1095-1103. doi: 10.1038/s41592-023-01820-3. Epub 2023 Mar 27.
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.
在行为相关的时间尺度上监测大量神经元群体的尖峰活动对于理解神经回路功能至关重要。与钙成像不同，电压成像需要千赫兹的采样率，将荧光检测降低到接近散粒噪声水平。高光子通量激发可以克服光子限制的散粒噪声，但光漂白和光损伤限制了同时成像神经元的数量和持续时间。我们研究了一种针对低双光子通量的替代方法，即低于散粒噪声限制的电压成像。该框架涉及开发具有改进尖峰检测的正向电压指示剂（SpikeyGi 和 SpikeyGi2）；一种用于在 0.4mm×0.4mm 视场中实现千赫兹帧率成像的双光子显微镜（'SMURF'）；以及一种用于从散粒噪声限制信号推断荧光的自监督去噪算法（DeepVID）。通过这些综合进展，我们在清醒行为小鼠中实现了超过 100 个高密度标记神经元的 1 小时以上的同时高速深层组织成像。这证明了一种可扩展的方法，用于在不断增加的神经元群体中进行电压成像。
