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.

Mok AT, Wang T, Zhao S, Kolkman KE, Wu D, Ouzounov DG, Seo C, Wu C, Fetcho JR, Xu C. A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging. eLight 2024;4:20. doi: 10.1186/s43593-024-00076-4.

 

Background: Large field-of-view (LFOV) deep imaging at single-cell resolution is essential for understanding brain-wide neuronal activity, yet traditional two-photon microscopy (2PM) is limited to superficial cortical layers, and three-photon microscopy (3PM) typically suffers from restricted imaging speed and area. To address these limitations, the authors designed an integrated system capable of deep, wide and high-resolution imaging across multiple cortical and subcortical regions.

 

Hypothesis: This study hypothesized that a combination of adaptive excitation, beamlet scanning and polygon-based high-speed scanning could enable simultaneous two- and three-photon imaging with large field-of-view and single-cell resolution at depths previously inaccessible to conventional systems.

 

Methods: The authors developed a custom multiphoton microscope (“DEEPscope”) that integrates adaptive excitation modules, a beamlet generation delay line and polygon-galvo scanners. The setup incorporated a vDAQ and used ScanImage for synchronized signal acquisition and virtual channel processing. Motion correction and neuron segmentation were performed with Suite2p, while three-dimensional reconstructions were generated in Imaris. Animal imaging experiments were conducted in awake transgenic mice and anesthetized zebrafish.

 

Results: The system achieved a 3.23-mm field of view with single-cell resolution, enabling imaging of cortical layer 6 and hippocampal neurons through intact cortex. It recorded activity from 917 neurons at 600 µm depth and from over 4,500 neurons during dual two- and three-photon imaging. Whole-brain zebrafish imaging resolved cellular nuclei to depths exceeding 1,090 µm.

 

Conclusions: This platform demonstrates a powerful, scalable approach for large-scale, deep and high-speed multiphoton imaging, advancing system-level investigations of neural circuitry.