A research team from the School of Engineering at The Hong Kong University of Science and Technology (HKUST) has achieved a major breakthrough in brain imaging by developing the world's first technology to capture high-resolution images of the brain of awake experimental mice in a nearly non-invasive manner. By eliminating the need for anesthesia, this innovation enables scientists to study brain tissue in its fully functional state. The advancement promises deeper insights into human brain function in both healthy and diseased conditions, opening new frontiers in neuroscience research.
The human brain is extraordinarily complex, and scientists have long sought to uncover its functions through brain imaging technologies. However, existing methods, such as magnetic resonance imaging (MRI), electroencephalography (EEG), computed tomography (CT), and positron emission tomography (PET), are limited in their ability to reveal the fine structural and functional details of brain activity.
Mice are widely used as model organisms to study treatments for neurological disorders such as Alzheimer's, Huntington's disease, and epilepsy, as well as therapies for various cancers and vaccine efficacy, due to their close genetic and biological similarity to humans. However, anesthesia profoundly alters blood circulation, glial cell morphology, and neuronal activity, leading to less reliable experimental results than those obtained from awake animals. Moreover, natural movements in awake mice often blur scanned images, hindering observation of the brain's fine structures.
The new technology, dubbed Multiplexing Digital Focus Sensing and Shaping (MD-FSS), was developed by a team led by Prof. QU Jianan, Professor of the Department of Electronic and Computer Engineering (ECE) of the School of Engineering. This innovation builds upon Prof. Qu's earlier work, Analog Lock-in Phase Detection Focus Sensing and Shaping (ALPHA-FSS), published in Nature Biotechnology in 2022. ALPHA-FSS achieved subcellular resolution in brain imaging using three-photon microscopy. Despite its high accuracy and high correction order, its scanning speed was too slow to capture high-quality images of brain issues of awake animals, where natural movements caused blurring. Furthermore, the skull's thickness and density strongly absorb and scatter incoming light, severely limiting the ability of two-photon microscopy to penetrate it. Even in superficial brain regions, image quality is degraded, resulting in poor imaging performance.
Sharper Images, Speeds Increased by Ten Times
To address these challenges, the team developed MD-FSS, which drastically accelerates the measurement of point spread function (PSF)-the three-dimensional image of a point-like object under the microscope. This groundbreaking method directs multiple spatially separated weak laser beams alongside a strong primary beam to generate nonlinear interference within the brain. Each beam is encoded at a unique frequency and carries distinct spatial information. Through parallel decoding via digital phase demodulation - a powerful technique for extracting faint signals from noisy backgrounds - the system achieves PSF measurements in less than 0.1 seconds, more than tenfold faster than prior methods, while tracking dynamic brain activity and producing sharp, precise images.
The resolution of multiphoton microscopy is hundreds to thousands of times higher than that of conventional methods such as EEG and CT, allowing for the observation of individual neurons, immune cells, and even the finest capillary structures and their functions. By integrating MD-FSS with multiphoton microscopy to develop the "Adaptive Optics Three-photon Microscopy", the research team demonstrated the technology's capability to track functional changes in brain immune cells, measure blood flow in the smallest cerebral vessels, monitor neuronal activity during cognitive and sensory processing, and capture interactions between brain cells and vasculature.
Prof. Qu said, "Such detailed, near noninvasive, and real-time observations in awake animals were previously impossible. With the rapid aberration-correction capability of this novel adaptive optics technology, high-quality imaging is now achievable without injuring the subject's brain. We can now capture the neuronal, glial, and vascular dynamics at subcellular resolution in their natural physiological state - free from the confounding effects of anesthesia. This breakthrough opens entirely new avenues for understanding brain function in both health and disease."
A Scalable Platform for Future Neuroscience
MD-FSS is engineered for future scalability. The current system, using eight beams for PSF measurement, can be expanded to dozens or even hundreds, enabling faster and broader imaging as advances in light-control technologies continue to emerge.
Prof. Qu added, "Our latest work represents far more than an incremental improvement. We now have a versatile platform that can be scaled for faster imaging, expanded into larger brain regions, and integrated with functional assays. This will empower neuroscientists to investigate rapid brain events, complex network interactions, and disease progression in ways that were previously technically unattainable - opening the door to transformative discoveries in learning, memory, mental health, and neurological disorders."
The research findings were recently published in Nature Communications in a paper titled "Rapid Adaptive Optics Enabling Near-Noninvasive High-Resolution Brain Imaging in Awake Behaving Mice". Prof. Qu Jianan and ECE PhD graduate Dr. QIN Zhongya are the co-corresponding authors. ECE PhD students SHE Zhentao and FU Yiming are the co-first authors.
