Imaging Deep and Distributed Neural Circuits with a Large Field-of-View Multiphoton Microscope

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.