Multibeam continuous axial scanning two-photon microscopy for in vivo volumetric imaging in mouse brain

This study presents an alternative approach for two-photon volumetric imaging that combines multibeam lateral scanning with continuous axial scanning using a confocal spinning-disk scanner and an electrically focus tunable lens. Using this proposed system, the brain of a living mouse could be imaged at a penetration depth of over 450 μm from the surface. In vivo volumetric Ca²⁺ imaging at a volume rate of 1.5 Hz within a depth range of 130-200 μm, was segmented with an axial pitch of approximately 5-µm and revealed spontaneous activity of neurons with their 3D positions. This study offers a practical microscope design equipped with compact scanners, a simple control system, and readily adjustable imaging parameters, which is crucial for the widespread adoption of two-photon volumetric imaging.