Integrated two-photon and optoacoustic microscopy for functional neuroimaging.

Understanding the interaction between cerebral vasculature and neurons is critical for studying neurovascular processes and their roles in brain function and neurological disorders. Existing functional neuroimaging approaches face trade-offs between resolution, penetration depth, and spatiotemporal alignment, limiting their ability to comprehensively image neurovascular anatomy and function in vivo. To address this challenge, we developed a dual-modality system that combines optical-resolution optoacoustic microscopy with two-photon fluorescence microscopy. The system enables imaging of microcapillaries with submicron resolution at up to 140 μm depth and neurons beyond 300 μm depth in the mouse cortex, thus providing complementary information. Using a semi-simultaneous acquisition protocol, the system alternately captures data across time and depth planes, ensuring spatiotemporal alignment, minimizing motion artifacts, and enabling robust co-registration of multimodal datasets for comprehensive studies of neurovascular coupling in health and disease.