Linear and Nonlinear Optical Imaging Enable Large Area Brain Morpho-Functional Imaging

High-resolution microscopy methods based on nonlinear optical technique find numerous applications in brain imaging. Functional or structural information can be gained with different implementations. In this work, large area reconstructions are obtained using a mesoscale light sheet system for structural analysis and a light sheet two-photon microscope for functional information. The mesoscale methodology developed allows analyzing the cytoarchitecture of the human brain in three dimensions at high resolution.

The combination of experimental protocols, based on optical tissue clearing and autofluorescence enhancement, with an automated software analysis enable to expand the histological studies to the third dimension. Functional imaging has been used to investigate whole organs, like zebrafish larval brain activity, using standard scanning or light sheet two-photon illumination. Both modalities are capable of sampling whole brain with single cell resolution, with light sheet imaging being capable to perform high-rate volumetric imaging, allowing to map in real time whole-brain calcium dynamics not affected by undesired visual stimulation artifacts, as occurring in one photon excitation fluorescence microscopy. The use of two photon optogenetics stimulation coupled to two photon light sheet imaging, finally enables closed loop experiments, with the assistance of AI, in order to perform tasks that are not doable under human guidance, such as functional connectivity reconstruction in short times.