Researchers have developed a revolutionary two-photon fluorescence microscope that captures neural activity at high speed and cellular resolution, providing unprecedented insight into how the brain works. This new approach, which produces images faster than traditional methods and causes less damage to brain tissue, could revolutionise our understanding of how neurons communicate in real time and potentially provide a breakthrough in treating neurological diseases such as Alzheimer’s and Parkinson’s.

A breakthrough in high-speed brain imaging

Researchers have developed a new two-photon fluorescence microscope that captures high-speed, cell-resolution images of neuronal activity. By capturing images much faster and with less damage to brain tissue than traditional two-photon microscopy, the new approach could provide a clearer picture of how neurons communicate in real time and lead to new insights into brain function and neurological diseases.

Real-time neural dynamics and brain function

“Our new microscope is ideal for studying the dynamics of neural networks in real time, which is critical for understanding basic brain functions such as learning, memory, and decision making,” said research team leader Weijian Yang of the University of California, Berkeley, Davis. “For example, researchers could use it to observe neural activity during learning and better understand the communication and interaction between different neurons during this process.”

INSIDE Optical In Optica Publishing Group’s high-impact research journal, researchers describe a new two-photon fluorescence microscope that incorporates a novel adaptive sampling scheme and replaces traditional point illumination with linear illumination. They show that the new method enables in vivo imaging of neuronal activity in the mouse cerebral cortex and can acquire images ten times faster than traditional two-photon microscopy, while also reducing laser power to the brain by more than tenfold.

“By providing a tool that can observe neuronal activity in real time, our technology can be used to study the pathology of diseases at the earliest stages,” said Yunyang Li, first author of the paper. “This could help researchers better understand and more effectively treat neurological diseases such as Alzheimer’s, Parkinson’s, and epilepsy.”

High-speed imaging with less damage

Two-photon microscopy can image deep into a scattering tissue, such as the mouse brain, by scanning a small spot of light across the entire sample area to excite fluorescence and then collecting the resulting signal from spot to spot. This process is then repeated to capture each frame of image. Although two-photon microscopy provides detailed images, it is slow and can damage brain tissue.

In the new study, the researchers aimed to overcome these limitations with a new sampling strategy. Instead of using a single spot of light, they use a short line of light to illuminate specific parts of the brain where neurons are active. This makes it possible to cover and image a larger area at once, which significantly speeds up the image acquisition process. Furthermore, since it images only the neurons of interest, not the background or inactive regions, the total light energy entering the brain tissue is reduced, reducing the risk of potential damage. They call this scheme adaptive sampling.

Advanced methods of target visualization

The researchers achieved this by using a digital micromirror device (DMD)—a chip containing thousands of tiny, individually controllable mirrors that dynamically shape and control the light beam, allowing precise targeting of active neurons. They achieved adaptive sampling by turning individual DMD pixels on and off to match the neuronal structure of the brain tissue being imaged.

The researchers also developed a technique to use DMDs to simulate high-resolution point scanning. This allows high-resolution images to be reconstructed using a fast scan and provides a fast way to identify neural regions of interest. This is critical for higher-speed imaging with short-line excitation and an adaptive sampling scheme.

“These advances, each important in their own right, come together to create a powerful imaging tool that greatly improves the ability to study dynamic neural processes in real time and reduces the risk to living tissue,” Yang said. “Importantly, our technique can be combined with other techniques, such as beam multiplexing and remote focusing, to further increase imaging speed or to obtain a 3D image.”

Capturing neural activity at unprecedented speed

The researchers demonstrated using the new microscope to image calcium signals—indicators of neuronal activity—in living mouse brain tissue. The system captured these signals at 198 Hz, much faster than traditional two-photon microscopes and demonstrates its ability to track fast neural events that might be missed by slower imaging methods.

They also showed that the adaptive line stimulation technique combined with advanced computational algorithms allows the activity of individual neurons to be isolated, which is important for accurately interpreting complex neural interactions and understanding the functional architecture of the brain.

Future improvements and programs

Next, the researchers are working to integrate voltage-imaging capabilities into the microscope to provide a direct and extremely fast readout of neuronal activity. They also plan to use the new method for real-world neuroscience applications, such as observing neural activity during learning and studying brain activity in disease states. They also aim to increase the microscope’s usability and reduce its size, expanding its use in neuroscience research.