A Large Field-of-View Two- and Three-Photon Microscope for High-Resolution Deep Tissue Imaging.

Two-photon microscopy has been engineered to image large populations of neurons in vivo. Three-photon microscopy has achieved a greater imaging depth. However, the attempt to increase its field of view has been hindered by its lower repetition rate. The key to overcoming this challenge is to engineer a scanning scheme that optimized each laser pulse for neuron excitation. We adopted an adaptive excitation scheme that scans solely the region of interest, minimizing wasted excitation pulses. Furthermore, we developed a multi-focus scanning method that increases both scanning speed and laser repetition rate. For the first time, we demonstrated three-photon calcium imaging of neurons within a ∼3.5mm diameter field-of-view at a 4Hz frame rate in the deepest cortical layers of mouse brains while preserving high spatial resolution. By reducing the three-photon imaging power, we achieved simultaneous multi-plane imaging with two- and three-photon techniques in both the superficial and deep cortical layers. The demonstrated adaptive scanning module can be integrated into multi-photon microscopes for large-field-of-view imaging, critical for system-level neural circuit research.