BME PhD Dissertation Defense - Grace Ye

Title: “Ultra-Fast Two-Photon Microscope for Population Neuronal Voltage Imaging”

Advisory Committee: Xue Han, PhD – BME (Chair) Jerry Chen, PhD – Biology, BME (Advisor) Jerome Mertz, PhD – BME, ECE, Physics Michelle Sander, PhD – ECE, BME, MSE Lei Tian, PhD – ECE, BME

Abstract: Understanding the complexity of neural networks in vivo requires simultaneous recordings of neuronal populations with action potential resolution. Genetically-encoded voltage indicators (GEVIs) that feature fast dynamics provide direct and reliable optical readouts of neural activity. The fast dynamic of spike-evoked GEVI responses necessitates the development of kilohertz-rate two-photon (2P) imaging systems. Existing high-speed 2P microscopes scan restricted field of views (FOVs), limiting the number of simultaneously imaged neurons.

We developed an Ultra-Fast 2-Photon (UF2P) microscope which is capable of full-frame high-speed imaging of a 400 µm x 400 µm FOV of up to ~300 µm deep in mouse cortex at a kilohertz scan rate. A combination of temporal and spatial multiplexing was applied in the system to generate eight parallel beams. A 920 nm-wavelength, 31.25 MHz repetition rate laser was used to deliver laser pulses that were temporally multiplexed into four interleaved beamlets. Emitted photons were de-multiplexed through digital temporal gating. Each beamlet was spatially multiplexed into beams that were spaced 200 µm apart at sample to avoid crosstalk introduced by light scattering in deep tissue. Photons from spatially multiplexed beams were resolved by the multi-anode photomultiplier tube.

The UF2P microscope was applied along with a novel positive-going voltage indicators with improved spike detection (SpikeyGi and SpikeyGi2) for in vivo voltage imaging in mouse cortex and a self-supervised denoising algorithm model (DeepVID) was trained for reducing shot noise in low photon-flux condition. Through combining all techniques above, simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons was achieved in awake behaving mice for over an hour with minimal effects on photobleaching, thermal effects, and photodamage during this period of time. The system demonstrates the capability for sustained voltage imaging across large neuronal populations.