Researchers have created a new two-photon fluorescence microscope that can quickly capture images of brain cell activity with high precision. This innovative method allows for faster imaging and less damage to brain tissue compared to conventional two-photon microscopy. As a result, it could offer a better understanding of how neurons communicate in real-time, potentially leading to new knowledge about brain functions and neurological disorders.
Researchers have created a new two-photon fluorescence microscope that can quickly capture images of brain cell activity with high precision. This innovative method allows for faster imaging and minimizes harm to brain tissue compared to traditional two-photon microscopy. Consequently, it could provide improved insights into the way neurons communicate in real time, potentially leading to new understandings of brain function and neurological disorders.
“Our new microscope is perfect for investigating the dynamics of neural networks in real time, which is essential for grasping core brain functions like learning, memory, and decision-making,” stated Weijian Yang, the leader of the research team at the University of California, Davis. “For instance, researchers can utilize it to watch neural activity during learning, gaining a better understanding of how various neurons interact and communicate in this process.”
In the journal Optica, published by Optica Publishing Group, the researchers detail the new two-photon fluorescence microscope, which features a novel adaptive sampling technique and replaces the conventional point illumination with line illumination. The study demonstrates that this new method allows for in vivo imaging of neuronal activity in a mouse cortex at speeds ten times faster than traditional two-photon microscopy while also reducing the laser’s effect on the brain by more than ten times.
“With a tool that can observe neuronal activity in real time, our technology stands to contribute to the study of disease pathologies from their earliest stages,” mentioned Yunyang Li, the primary author of the study. “This could enhance researchers’ understanding and treatment of neurological diseases like Alzheimer’s, Parkinson’s, and epilepsy.”
High-speed imaging with reduced harm
Conventional two-photon microscopy can penetrate deep into scattering tissues, like a mouse brain, by scanning a small light point across the entire area to stimulate fluorescence, capturing the emitted signal point by point. This process is repeated to form each image frame. While two-photon microscopy creates detailed images, it is slow and can be damaging to brain tissue.
In this new study, the researchers sought to address these challenges with an innovative sampling strategy. Instead of relying on a single point of light, they use a short line of light to stimulate designated areas of the brain where neurons are active. This method allows for the simultaneous excitation and imaging of a larger area, significantly speeding up the imaging process. Furthermore, it focuses only on active neurons rather than the surrounding inactive areas, reducing the total light energy deposited to the brain and consequently lowering the risk of damage. This technique is referred to as adaptive sampling.
The team achieved this by utilizing a digital micromirror device (DMD), which consists of thousands of tiny mirrors that can be independently controlled to dynamically shape and direct the light beam, targeting active neurons precisely. They implemented adaptive sampling by selectively turning DMD pixels on and off, which adjusts based on the neuronal structure of the brain tissue being observed.
Additionally, the researchers devised a technique to use the DMD to simulate high-resolution point scanning. This setup allows for the reconstruction of high-resolution images from rapid scans, providing a swift method to pinpoint neuronal regions of interest. This step is crucial for subsequent high-speed imaging using the short-line excitation and adaptive sampling strategy.
“These advancements — each vital on its own — combine to form a powerful imaging tool that significantly enhances our ability to study dynamic neural processes in real time, while reducing potential risks to living tissue,” Yang explained. “Importantly, our technique can be integrated with other methods like beam multiplexing and remote focusing to further boost imaging speed or enable volumetric 3D imaging.”
Capturing neural activity
The researchers showcased the new microscope by imaging calcium signals, which indicate neural activity, in live mouse brain tissue. The system was able to capture these signals at a rate of 198 Hz, significantly faster than traditional two-photon microscopes, and capable of detecting rapid neuronal activities that slower methods might miss.
Moreover, they demonstrated that the adaptive line-excitation technique, combined with advanced computational algorithms, allows for the isolation of individual neurons’ activities. This capability is crucial for accurately interpreting complex neural interactions and understanding the functional structure of the brain.
Moving forward, the researchers are working to add voltage imaging capabilities to the microscope for an immediate and highly rapid readout of neural activity. They also plan to apply the new method in real neuroscience scenarios, such as monitoring neural activity during learning and examining brain function in disease conditions. Additionally, there are efforts to enhance the microscope’s usability and decrease its size to make it more practical for neuroscience research.
