By Beth Miller
The brain relies on real-time delivery of oxygen and nutrients through its microvasculature, which threads through neural tissue like electrical wires. While modern imaging technologies allow researchers to follow the activity of individual neurons in the brain, they are not yet advanced enough to dissect the microvascular function at a comparable spatial scale. This gap hinders our understanding of cerebral small vessel disease and its contributions to cognitive impairment and dementia.
To address this challenge, a team of researchers at Washington University in St. Louis and Northwestern University, led by Song Hu, professor of biomedical engineering in the McKelvey School of Engineering, has developed super-resolution functional photoacoustic microscopy (SR-fPAM). By tracking the movement and oxygenation-dependent color change of red blood cells, SR-fPAM allows researchers to image blood flow and oxygenation at single-cell resolution in the mouse brain, which bridges a critical gap in functional microvascular imaging and could provide new insight into microvascular health and disease, such as stroke, vascular dementia and Alzheimer's disease.
Results of the research were published March 3, 2026, in Light: Science & Applications.
Red blood cells, which are abundant in blood vessels, naturally absorb light due to hemoglobin, the molecule responsible for oxygen transport. When illuminated with short laser pulses, hemoglobin generates ultrasound waves, a phenomenon known as the photoacoustic effect. While conventional photoacoustic microscopy can image blood vessels without labeling them, it does not provide single-cell resolution in 3D.
Hu's team addressed this limitation by developing a high-speed photoacoustic microscope capable of repeatedly imaging the same brain region at millisecond intervals, allowing them to trace red blood cell movement in single files through capillaries and in groups through larger vessels. By tracking these cells across sequential frames and accumulating their trajectories computationally, the researchers were able to reconstruct 3D microvascular structures at single-cell resolution.
"Similar to super-resolution fluorescence and ultrasound imaging, SR-fPAM leverages high-speed imaging to track dynamics and uses that information to identify features that are smaller than the conventional resolution limit," Hu said. "We condense multiple spatiotemporally acquired frames into a single one with substantially improved resolution."
In their experiments, SR-fPAM revealed how blood flow and oxygenation redistributed across 3D microvascular networks in the brain after an induced stroke. When a single microvessel was occluded, nearby vessels instantly changed their flow patterns, redirecting red blood cells to help sustain oxygen delivery to the affected tissue
"When one vessel is blocked, red blood cells take alternative routes to continue the flow and oxygen supply," Hu said. "Using SR-fPAM, we can observe not only structural changes in the 3D microvasculature, but also how fast red blood cells move, how their flow directions change, and how they release oxygen into the surrounding tissue in response to stroke-induced ischemia."
Looking ahead, Hu and his team want to pair SR-fPAM with two-photon microscopy so that we can simultaneously image both red blood cells and neurons at single-cell resolution.
"This would allow us to study how neurons and microvessels are spatiotemporally coordinated with each other and how their dynamic coupling gets disrupted in disease," Hu said. "It may also help us better interpret clinical neuroimaging techniques, such as functional MRI, which infers brain activity from vascular signals.
Hu said this work could have a significant translational impact.
"Cerebral small vessel disease is increasingly recognized as a leading cause of cognitive impairment and dementia, and WashU is at the frontier of this in both basic and clinical research," Hu said. "If we can better understand how microvascular oxygenation and flow change in the early stages of disease, it may help guide the development of early detection strategies and therapeutic interventions."
Zhong F, Wang Z, Lee Y, Han J, Sun N, Yang S, Ji S, Zhang HF, Sun C, Hu S. Super-resolution functional photoacoustic microscopy via label-free cell tracking. Light: Science & Applications. March 3, 2026, DOI: https://doi.org/10.1038/s41377-026-02235-3 .
This research was supported in part by the National Institutes of Health (NS099261, NS120481, NS125677, AG079503, P41GM135018), the National Science Foundation (202988), the Chan Zuckerberg Initiative Frontiers of Imaging Award, and Washington University's Imaging Sciences Pathway Fellowship. Work performed at the Center for Nanoscale Materials, a U.S. Department of Energy Office of Science User Facility, was supported by the U.S. DOE, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.
