Hyperspectral characterization of hemodynamic and metabolic responses in the mouse brain during oxygenation alterations

Hyperspectral imaging is a promising approach for characterizing chromophore-dependent contrast in living tissue. In this work, we report the in vivo validation of HyperProbe, a hyperspectral imaging system originally developed to support neuronavigation during human brain surgery and here tested in a mouse model. By precisely controlling the inhaled oxygen concentration, we induced transitions between normoxia, hyperoxia, and anoxia, and monitored the resulting hemodynamic and metabolic responses through hemoglobin and cytochrome signatures. Using full-spectrum acquisitions (375–1015 nm) combined with spectral unmixing, we derived spatially resolved maps that reflect relative changes in molecular abundances across the cortical surface. We also implemented a three-wavelength protocol that enabled faster tracking of oxygenation dynamics and vascular responses within the same field of view. These results demonstrate the ability of HyperProbe to detect oxygenation-driven variations in molecular contrast, supporting its future translation to neurosurgical applications.