Simultaneous head-mounted imaging of neural and hemodynamic activities at high spatiotemporal resolution in freely behaving mice

Exploring the relationship between neural activity and cerebral hemoglobin-oxygenation responses in freely behaving mice can advance our understanding of neurovascular coupling. Head-mounted microscopes enable neuroimaging in freely behaving mice; however, investigating neurovascular dynamics remains challenging because of a lack of hemodynamic information, especially blood oxygenation, or insufficient resolution. Here, we report a head-mounted microscope for neurovascular imaging that enables the simultaneous recording of neuronal burst firing and multiparametric hemodynamics such as vascular oxygen saturation at high spatiotemporal resolution. The 1.7-gram lightweight microscope integrates confocal fluorescence and photoacoustic microscopy, allowing recordings at 0.78 hertz with 1.5-micrometer lateral resolution across a field of view of 400 micrometers by 400 micrometers. We identified cell type–specific neurovascular responses to hypoxic challenges, observed active regulation of arterioles during sensory stimuli, and detected abnormal oxygen depletion and vasodilation preceding burst neuronal discharges in epileptic disorders. This technique provides valuable insights into neurovascular coupling and holds potential for studying the pathology of neurological brain diseases.