Multimodal 3-photon microscopy for deep-tissue analysis of blood oxygenation and neuronal activity – 3P-NEUROVASC

Functional hyperemia is the increase in oxygenated blood flow triggered by neuronal activation and generated by neurovascular coupling (NVC), an ensemble of pathways involving the interactions of neurons, astrocytes, endothelial cells, and contractile cells. fMRI (“BOLD”) signals probe changes in blood oxygenation with millimeter resolution and depend on the complex interplay of oxygen consumption, blood volume, and velocity changes that occur during NVC. Therefore, mapping red blood cell (RBC) flow, neuronal activation, and RBC oxygenation at the microscopic level is critical for understanding the relationship between local NVC and the fMRI signal. However, current microscopy methods for imaging RBC oxygenation in animal models are limited in spatial and/or temporal resolution and not easily compatible with simultaneous fluorescence-based imaging of neuronal activity. The goal of this project is to develop a novel form of advanced multimodal multiphoton microscopy that will allow simultaneous imaging of neuronal activity, blood flow dynamics, and RBC oxygenation with unique capabilities: millimeter imaging depth, micrometric 3D resolution, and microsecond pixel times. This new approach will be used to analyze NVC in model systems such as the mouse olfactory bulb. Our project is based on the implementation of simultaneous detection of three-photon (3P) fluorescence signals from calcium reporters and label-free third-order sum frequency generation (TSFG) signals reporting on RBC flow and oxygenation. TSFG using dual femtosecond excitation is a spectroscopic imaging modality recently introduced by us. TSFG is sensitive to haemoglobin absorption and is compatible with 3P microscopy laser technology. Multimodal 3P-TSFG microscopy will provide a groundbreaking approach for the analysis of brain physiology in intact tissue with cellular resolution. Our consortium is at the forefront of this new technological advance and its applications.