A large field of view two- and three-photon microscope for high-resolution deep tissue imaging

We have developed a two- and three- photon microscope that enables high-resolution imaging with a large field-of-view of ~3.5 mm diameter at > 1 mm depth of mouse brains.

1. Introduction

High spatial and temporal resolution imaging over a large field-of-view (FOV) deep within intact tissues is valuable for many biological fields such as neuroscience, immunology, and cancer biology. While large FOV two-photon microscopy (2PM) [1] has been successfully demonstrated in recording neural activities with up to 5 mm FOV, the penetration depth of two-photon (2P) imaging is limited to 600 to 700 µm in the intact mouse brain [2], restricted to the shallow cortical layers. In contrast, three-photon microscopy (3PM) has been shown to reliably image neurons in deep cortical layers, subplates, and subcortex [3]. In addition, a combination of 2PM and 3PM enabled neural imaging in the neocortex and the sub-cortical region simultaneously with a limited FOV [4] of ~ 0.5 x 0.5 mm².