New photoacoustic probes enable imaging of deep brain tissue, with the potential to report neuronal activity

To better understand the brain, we need new ways to observe its activity. This is at the heart of a molecular engineering project, led by two research groups at the European Molecular Biology Laboratory (EMBL), which has resulted in a new approach to creating photoacoustic probes for neuroscience applications. The results have been published in the journal Neuroscience. Journal of the American Chemical Society.

“Photoacoustics can capture images of the entire brain of a mouse, but we simply lacked the right probes to visualize the activity of a single neuron,” says Robert Prevedel, EMBL group leader and lead author of the paper. To overcome this technological challenge, he worked with Claire Deo, another EMBL group leader and lead author of the paper. She and her team specialize in chemical engineering.

“We were able to show that we can actually label neurons in specific areas of the brain with probes that are bright enough to be detected by our custom photoacoustic microscope,” Prevedel said.

Scientists can learn more about biological processes by tracking certain chemicals, such as ions or biomolecules. Photoacoustic probes can act as “reporters” for hard-to-detect chemicals by binding specifically to them. The probes can then absorb light when excited by lasers and emit sound waves that can be detected by specialized imaging equipment.

For neuroscience applications, however, researchers have so far been unable to design targeted reporters capable of visualizing brain functions suitable for photoacoustics.

Researchers have experimented with using synthetic dyes as photoacoustic indicators of neuronal activity, but it has been difficult to control where the dye goes and what might be labeled. Proteins have proven particularly useful as probes for labeling specific molecules, but have not yet led to effective photoacoustic probes for monitoring neuronal activity throughout the brain.

“In our case, we took the best of both worlds, combining a protein with a rationally designed synthetic dye, and we can now label and visualize neurons in specific regions of interest,” said Alexander Cook, first author of the study and a predoctoral researcher in the Deo group. In rational design approaches, researchers use existing knowledge and principles to build molecules with desired properties, rather than blindly making and testing random compounds.

“Furthermore, we are not just talking about a static observation, but this probe shows a reversible and dynamic response to calcium, which is a marker of neuronal activity,” Cook added.

According to Deo, this technological development faced a major challenge: because photoacoustic probes had not been studied in depth, researchers did not have the means to evaluate the probes they were building.

The project thus began with Nikita Kaydanov, co-author of the study and predoctoral researcher in the Prevedel group, who custom-built a spectroscopy setup.

“There is no commercial device that can measure photoacoustic signals from a probe in test tubes or cuvettes, so we had to build one,” Kaydanov says. “We created our own photoacoustic spectrometer to evaluate and optimize the probes.”

“This allowed us to evaluate and characterize the different probes that we made to evaluate several things,” Deo said. “Did they produce a detectable photoacoustic signal? Are they sensitive enough? That’s how we deduced the next steps.”

But the researchers didn’t want to stop at producing working probes in a bottle. They then wanted to see how the probes worked in practice. They found a way to introduce the probes into the brain of a mouse and were able to detect photoacoustic signals from neurons in the targeted brain regions.

“While we are excited about the progress, we must be clear: this is only the first generation of these probes,” Deo said. “While they offer a very promising approach, we still have a lot of work to do, but this is a good first demonstration of what this system can do and the potential it has to better understand how the brain works.”

In fact, these next steps include improving the dye delivery system and confirming the ability to use them for dynamic imaging inside cells.

“One of the great things about EMBL is that it brings together so many people with different skills,” Prevedel said. “We are both developers in our own way: my group works more on instrumentation, and Claire’s on molecular tools. And by combining that with neuroscientists who then actually test the tools, we have a special and unique way of doing research that is only possible at EMBL.”