Today, researchers can image the body with a variety of techniques, from using traditional light and lasers to magnetic resonance and ultrasound. Junjie Yao, an imaging scientist at Duke University, develops photoacoustic imaging techniques for biomedical and clinical research. In one of these approaches, called photoacoustic tomography (PAT), Yao and his team use lasers to excite biological molecules so that they produce ultrasonic waves that their tomography system can detect.
Collaborating with Wei Yang, a neuroscientist also at Duke University, Yao sought to use PAT as a way to study the glymphatic system, the process of waste removal in the brain. This process is separate from the traditional lymphatic system, which removes waste throughout the rest of the body. Instead, in the glymphatic system, blood pressure from the vascular system pushes cerebrospinal fluid (CSF) around the spaces surrounding the blood vessels, picking up and removing metabolic byproducts and neuronal debris through diffusion.
However, researchers have struggled to study the glymphatic system in real time and in detail because of limitations in either resolution, penetration, or the overall field of view. For Yang and Yao, PAT presented an opportunity to noninvasively monitor the movement of the CSF and how changes to vascular dynamics influenced this. Coupled with another microscopy technique to aid in imaging the movement of blood throughout the brain, the researchers developed a novel imaging method that could help scientists better understand how changes in the brain and its blood supply affect this vital system.
3D-PAULM, a technique developed by Duke University researchers, reveals the brain's vasculature (blue/green/yellow) and glympatic system (red/orange/yellow) simultaneously.
Nanchao Wang and Junjie Yao, Duke University
As a metabolically active organ, the brain produces byproducts and debris that, if not removed, would build up and cause damage. “It's like your city’s garbage recycling system is on strike, or it's not working. So, you will accumulate a lot of waste around the neighborhood, and this is going to get worse and worse over time,” Yao said. Previously, researchers showed that dysfunction to the glymphatic system negatively impacts neurological conditions like Alzheimer’s disease, Parkinson’s disease, and traumatic brain injury.1-3
In contrast to many other imaging methods, PAT can noninvasively view both the glymphatic and vascular systems to study how blood pressure pushes the CSF through the brain. Normally, the CSF is transparent with few molecules that produce ultrasonic waves, but Yao and his team introduced infrared tracer molecules directly to the glymphatic system to provide the necessary contrast to visualize the system better. This addition addressed the invisibility problem, but PAT alone does not have the spatial resolution to assess the entire brain vasculature. So, the duo partnered with Pengfei Song, a biomedical engineer using ultrasound localization microscopy (ULM) at Duke University, whom Yao had worked with separately. “[It was] very exciting to see old friends for a new [project],” he added.
With Song’s expertise, the team used a customized system to not only pick up ultrasound signals from Yao’s laser-excited molecules but also to transmit and detect its own ultrasound signals. Leveraging this ability, the researchers introduced gas-filled microbubbles into the vascular system of mice, which responded to ultrasound at a different frequency than the tracers in the glymphatic system, allowing the scientists to detect them simultaneously.
Yet, before the researchers could successfully study the vasculature and glymphatic system, they had to cross one final hurdle: speed. “It's a dynamic system. We need to be fast enough to capture how the blood is moving, how the glymphatic system is moving,” Yao said.
To capture this flow around the brain, the researchers developed hardware that could synchronize the PAT and ULM outputs as well as a computational model to process these signals in real time. Combining these two technologies in what the team dubbed 3D-PAULM, Yao said, overcame this final obstacle. “It's like now we can see both the highways of the city and also the local streets of the city.”
Yao, Yang, and Song overcame several biological and technical challenges to image the brain’s glymphatic system in high resolution and in real time. The 3D-PAULM technique combines photoacoustic and ultrasound localization microscopy to reveal the brain’s vasculature (cooler colors indicate greater depth) and CSF of the glymphatic system (more yellow indicates greater signal intensity).
Nanchao Wang and Junjie Yao, Duke University
The 3D-PAULM images recreated the brain’s vasculature, showing deeper vessels in blues and greens and vessels closer to the surface in red and orange. The researchers overlaid the red, orange, and yellow images of the glymphatic CSF that surrounded these vessels. Recently, the team showed how the technique could track blood flow and the glymphatic system simultaneously.4 They also used it to identify changes in these processes in models of stroke, aging, and anesthesia.4
Yao said that, as an imaging scientist who has been developing these techniques for a decade, his excitement gets refreshed every day as he and his team push technological boundaries. When he saw the first images from 3D-PAULM, he recalled thinking, “This is great. Can we do more? Can we study a little bit more?”
Indeed, he and his collaborators have been excited to have other scientists reach out to them about taking the technique further, processing the data differently, or even using the findings to help train AI models. Yao said that he will be most happy to see the group’s findings and their technique go beyond foundational research and help improve treatments for stroke or promote brain health. Additionally, he said that 3D-PAULM can also have applications beyond the brain, as he and his team are currently collaborating with scientists to study the tumor microenvironment.
Junjie Yao is a cofounder and consultant for Lumius Imaging Inc. The company did not fund the current work and is not currently pursuing commercialization of 3D-PAULM.
- Da Mesquita S, et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease. Nature. 2018;560(7717):185-191.
- Bolte AC, et al. Meningeal lymphatic dysfunction exacerbates traumatic brain injury pathogenesis. Nat Commun. 2020;11(1):4524.
- Ding X-B, et al. Impaired meningeal lymphatic drainage in patients with idiopathic Parkinson’s disease. Nat Med. 2021;27(3):411-418.
- Wang N, et al. Noninvasive whole-brain imaging of glymphatic dynamics. Sci Adv. 2026;12(25):aee4926.
