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.

Simultaneously monitoring cortex-wide microvascular dynamics, blood oxygenation metabolism, and neuronal activities with high spatiotemporal resolution is essential for understanding neurovascular coupling (NVC). However, it remains a challenge for existing neuroimaging tools. Here, we report a linear transducer array–based hybrid microscope (LiTA-HM) that integrates photoacoustic microscopy and confocal fluorescence microscopy to achieve simultaneous neurovascular imaging with a field of view (FOV) of 6 millimeters by 5 millimeters, a spatial resolution of ~6 micrometers, and a temporal resolution of 0.8 seconds. We design an array of eight miniature transducers to enlarge FOV and optimize a polygon mirror–based ultrafast scanner to achieve real-time imaging. Proof-of-concept imaging experiments were conducted on awake mice. Different couplings between thousands of neuron somas and vessel branches down to capillaries were observed in hypoxia and anesthesia experiments. Spatiotemporal correlations and functional connectivity in NVC were uncovered for spreading depolarization waves occurred in a drug-induced epilepsy experiment. We expect that LiTA-HM will enable broad applications of neuroimaging.

A widely used cerebrovascular stimulus and common pathophysiologic condition, hypercapnia is of great interest in brain research. However, it remains controversial how hypercapnia affects brain hemodynamics and energy metabolism. By using multi-parametric photoacoustic microscopy, the multifaceted responses of the awake mouse brain to different levels of hypercapnia are investigated. Our results show significant and vessel type-dependent increases of the vessel diameter and blood flow in response to the hypercapnic challenges, along with a decrease in oxygen extraction fraction due to elevated venous blood oxygenation. Interestingly, the increased blood flow and decreased oxygen extraction are not commensurate with each other, which leads to reduced cerebral oxygen metabolism. Further, time-lapse imaging over 2-hour chronic hypercapnic challenges reveals that the structural, functional, and metabolic changes induced by severe hypercapnia (10% CO₂) are not only more pronounced but more enduring than those induced by mild hypercapnia (5% CO₂), indicating that the extent of brain’s compensatory response to chronic hypercapnia is inversely related to the severity of the challenge. Offering quantitative, dynamic, and CO₂ level-dependent insights into the hemodynamic and metabolic responses of the brain to hypercapnia, these findings might provide useful guidance to the application of hypercapnia in brain research.

Providing the unique contrast of optical absorption and excellent scalability of spatial resolution and tissue penetration depth, photoacoustic imaging has emerged as an enabling technology for brain research.^1,2 Capitalizing on the optical absorption of hemoglobin, multiparametric photoacoustic microscopy (PAM) is uniquely capable of comprehensive characterization of the cerebral vasculature in vivo.³ Specifically, statistical, spectroscopic, and correlation analysis of the same dataset allows simultaneous quantification of the total concentration of hemoglobin ($C_{\mathrm{Hb}}$), oxygen saturation of hemoglobin (${\mathrm{sO}}_2$), and cerebral blood flow (CBF) at the microscopic level.⁴ Moreover, combing these hemodynamic measurements enables the quantification of the cerebral metabolic rate of oxygen.⁵

Traditional solutions for Ethernet transmission of data acquisition systems (DAQ) are mainly based on software transmission control protocol/internet protocol (TCP/IP) stack while its throughput is relatively low and hard to improve. A hardware TCP/IP stack that can greatly promote the Ethernet throughput for generic DAQ is presented. To reduce the memory overhead of FPGA in DAQ, a shared memory method based on parallel and pipeline operations is proposed so that it can realise multiple protocols with just 2 Kb RAM for receiving and sending respectively. Unlike software system, hardware stack is difficult to realise multiple data source transmission simultaneously since it is realised totally by logic circuits. Therefore, a data manage module is designed specially to manage the internal, software and hardware data and it can enhance the flexibility and practicability remarkably. Functional simulation shows that, under 50 MHz system clock, the maximum throughput is near 800 Mbps. A common Megabit Ethernet controller chip is applied in the board test. The results show that the maximum throughput of the whole system is 82.2 Mbps which is much better than designs based on the same or similar network controllers.