Adaptive Excitation Two-Photon Microscopy for Deep, Fast Voltage Imaging

Zhao S, Hebert E, Gruzdeva A, Mahishi D, Takahashi H, Lee S, Hao YA, Lin MZ, Yapici N, Xu C. Deep two-photon voltage imaging with adaptive excitation. Res Sq [Preprint] 2024 Dec 13:rs.3.rs-5434919. doi: 10.21203/rs.3.rs-5434919/v1.

Background: Optical monitoring of neuronal voltage dynamics enables the study of fast neural activity with subcellular resolution. However, two-photon microscopy (2PM) suffers from depth and speed limitations due to tissue heating and reduced photon flux. Existing methods either image superficial neurons or single cells in deeper layers, often requiring complex beam multiplexing or high laser powers that risk thermal damage.

Hypothesis: This study hypothesized that incorporating adaptive excitation into high-speed 2PM could achieve deep, simultaneous voltage imaging of multiple neurons in vivo while remaining below the tissue heating threshold.

Methods: The authors developed a dual-plane adaptive excitation two-photon microscope combining a polygon-galvanometer scanner with Pockels cell–based illumination control synchronized via an acquisition system and ScanImage. Data were acquired through ScanImage and processed with MATLAB, while system synchronization and digitization were coordinated through a vDAQ. Neurons expressing the genetically encoded voltage indicator ASAP5 were imaged in the visual cortex of awake mice.

Results: Adaptive excitation enhanced signal collection 40–50-fold compared to conventional imaging at identical power, enabling detection of supra- and subthreshold neuronal activities at depths up to 635 µm with signal-to-noise ratios above 9. Dual-plane imaging across 80–115 µm axial separations showed minimal crosstalk and synchronized subthreshold oscillations in deep layers.

Conclusions: Adaptive excitation permits noninvasive, high-speed, deep two-photon voltage imaging of multiple neurons, approaching the theoretical performance limit for this technique and offering straightforward implementation on standard microscopes.