The image shows neuronal calcium activity (green) and vascular hemoglobin concentration (red). A new microscopy technique developed at WashU can show how the brain’s red blood cells deliver oxygen to its neurons, a tool that could better enable research on stroke and dementia.
Image Credit: Song Hu/Washington University in St. Louis
Scientific Frontline: Extended "At a Glance" Summary: Integrated TPM-PAM Microscopy
The Core Concept: Integrated two-photon and photoacoustic microscopy (TPM-PAM) is a novel imaging platform that simultaneously captures single-neuron calcium activity alongside oxygen delivery from individual red blood cells in real time.
Key Distinction/Mechanism: Unlike functional MRI, which infers brain activity indirectly from blood oxygenation, or conventional microscopy that requires separate systems, TPM-PAM merges two technologies using an optically transparent acoustic sensor. This micro-ring resonator allows both light and sound waves to share the same space without compromising the optical resolution of two-photon microscopy or the oxygen-recording capabilities of photoacoustic microscopy.
Major Frameworks/Components:
- Two-Photon Microscopy (TPM): Utilizes fluorescent probes to image neuronal calcium activity at a cellular resolution.
- Photoacoustic Microscopy (PAM): Employs light-generated sound waves to measure blood flow and oxygenation dynamics within the microvasculature.
- Polymer Micro-Ring Resonator: An optically transparent acoustic sensor built on glass that converts ultrasound signals into measurable shifts in optical resonance without blocking the optical pathway.
- Neurovascular Coupling: The physiological mechanism linking localized neural activity to corresponding, immediate changes in cerebral blood flow and oxygen delivery.
Branch of Science: Biomedical Engineering, Neuroscience, Optics, and Photonics.
Future Application: This platform provides a direct method for probing the cause-and-effect relationship in neurovascular coupling, which could significantly enhance our understanding of metabolic failures in neurodegenerative diseases and stroke. Furthermore, establishing a precise cellular-scale baseline could improve the interpretation and accuracy of functional MRI signals in clinical settings.
Why It Matters: The brain relies on a tightly regulated energy supply, yet the precise cellular coordination between neurons and the microvessels that fuel them has remained difficult to observe directly. By successfully uniting two disparate forms of energy—light and sound—this innovation opens a new window into how cellular energy demands are met and how that relationship degrades during disease progression.
The brain is a black box of densely packed neurons, blood vessels, and immune cells, all locked away in a sealed skull. Song Hu of Washington University in St. Louis has dedicated his career to unlocking that puzzle box by developing new ways to “see” inside. His team’s newest innovation could give researchers a new view of how brain cells and blood vessels work together, as well as what happens when the coupling between the two breaks down.
Hu, a professor of biomedical engineering at WashU, and his team in the McKelvey School of Engineering have developed a new way to integrate two powerful techniques: two-photon microscopy and photoacoustic microscopy (TPM-PAM). This breakthrough will allow researchers to see in real time how the brain’s vasculature delivers oxygen to neurons. It could have profound implications for studying neurodegenerative diseases, stroke, and other conditions connected to neurovascular coupling.
“Using this technology gives us a better understanding of these disease mechanisms,” Hu said.
Hu and his team shared their results in the journal Nature Communications, detailing how they combined the two different forms of microscopy into a single system and successfully recorded, for the first time, single-neuron calcium activity alongside oxygen release from individual red blood cells in awake mice.
Watching Neuronal Activity and Oxygen Delivery in Action
Two-photon microscopy (TPM) is widely used to image neuronal activity in the brain, which is “lit up” by fluorescent probes.
The other technique, photoacoustic microscopy (PAM), uses light-generated sound waves to record blood flow and oxygenation. The brain's function is intricately linked to its vasculature. “Thinking” itself consumes energy, but without the view of oxygen delivery that PAM can provide, it is very difficult to understand the details of how the fuel pipeline works in relation to brain activity.
“That’s why it’s important to image both of them, to understand how neuronal activity consumes oxygen and how the vasculature changes its dynamics to meet the demand in real time,” Hu said.
With this new technique, Hu and his team were able to bring the two complementary views together at a cellular resolution for the first time.
“Combining the two for simultaneous imaging of neuronal activity and oxygen delivery at the single-cell level has never been demonstrated before,” Hu added.
To demonstrate the platform, the team imaged the brains of mice while stimulating their whiskers, tracking neuronal calcium activity alongside changes in blood oxygen delivery. The researchers also used tightly focused laser pulses to block a single capillary or stimulate a single neuron; they then followed the resulting changes in nearby neurons and red blood cells. Together, these experiments show how TPM-PAM can combine precise, cell-scale interventions with simultaneous measurements of neuronal activity and oxygen delivery, allowing researchers to move beyond observing correlations and begin probing cause and effect.
Although demonstrated so far only in animal models, the platform could help reveal how brain cells and microvessels coordinate energy use and how that relationship is disrupted in stroke and neurodegenerative diseases.
This platform could also inform clinical imaging. Functional magnetic resonance imaging (fMRI) does not directly record neurons; rather, it infers changes in brain activity from blood oxygenation and flow. A clearer cellular-scale picture of neurovascular coupling could help researchers interpret these indirect signals much more accurately.
“If you can better understand neurovascular coupling, you will make this inference process more accurate,” Hu said.
Making Room for Light and Sound
The technical challenge was solved with help from engineering colleagues at Northwestern University.
Light and sound waves do not usually play well together.
“If you want to integrate the two, you have to make the two forms of energy share the same space, and this is not easy,” Hu said.
Conventional PAM systems use ultrasound detectors that block the optical path or require optical lenses that sacrifice two-photon resolution and light collection. The team’s solution is an optically transparent acoustic sensor built from a polymer microring resonator on a piece of glass. When ultrasound impinges on the resonator, it slightly deforms the ring and shifts its optical resonance, converting the acoustic signal into a measurable change in light. Simultaneously, the transparent sensor allows TPM’s excitation and fluorescence light to pass through with minimal interference.
“It’s not compromising either of the two imaging technologies,” Hu said.
Funding: This work was supported by the National Institutes of Health (R01 NS120481, AG079503, and NS125677 to S.H.; P41 GM135018 to H.F.Z. and C.S.) and the Washington University Imaging Sciences Pathway Fellowship (to J.H.).
Published in journal: Nature Communications
Title: Integrated two-photon and photoacoustic microscopy for single-cell neurometabolic imaging
Authors: Jiaxiao Han, Youngseop Lee, Ziang Feng, Yue Wu, Zhuoying Wang, Allison Martinez Mejia, Adam Bauer, Manu Goyal, Jin-Moo Lee, Peinan Zhao, Hao F. Zhang, Cheng Sun, and Song Hu
Source/Credit: Washington University in St. Louis (McKelvey School of Engineering) | Leah Shaffer
Edited by: Scientific Frontline
Reference Number: eng091026_01
