Implementation and use of Large Field-of-view Two-photon Microscopy for in vivo imaging

Abstract

Studying brain network, which defines how neurons and neural networks process information, is crucial to understand how the brain functions. The study of brain network connectivity has recently received a lot of interest thanks to the use of two-photon microscopy. In contrast to conventional whole-brain functional medical imaging techniques like PET or fMRI, two-photon microscopy allows sub-cellular level in vivo imaging of the dynamics in individual neurons and identifying the interaction between cells and blood vessels with its high resolution. Conventional two-photon microscopy, however, has a small field-of-view (FOV) that can only cover part of a mouse brain. To overcome this, a large FOV two-photon microscopy with a FOV of 16x8mm2 was developed using a dual objective lens system so that the whole mouse brain cortex could be imaged at once. In this study, in vivo imaging was performed using the home-built large FOV two-photon microscopy to establish an in vivo research environment feasible for blood flow and lymphatic circulation study. It was demonstrated that imaging the entire structure in a wide FOV and selecting a region of interest (ROI) to observe the dynamics in specific ROI. Additionally, to quantify blood flow velocity in the ROI, OSIV (optical speckle image velocimetry), which applied only to simple vascular structures, was enhanced to be applicable to complex vascular structures.