[Enlarge image]In adaptive line-excitation, a high-resolution video of the neuronal activity is first acquired through an equivalent point-scanning strategy. The regions of interest (ROIs) are then segmented and binarized into a mask and loaded to a digital micromirror device (DMD). The DMD-diffracted beam, carrying ROI information, illuminates the corresponding sample part.
Traditional two-photon microscopy involves raster scanning a single diffraction-limited point of light across the entire sample area to construct an image.1 While this approach is effective for high-resolution imaging, it is inherently slow and may expose the brain tissue to excessive light, increasing the risk of photodamage. Beam multiplexing can increase imaging speed,2 but it also exposes the brain to higher laser power and thus increases the risk of thermal damage. To address these limitations, we developed a novel method that significantly enhances imaging speed while minimizing the risk of tissue damage.3
At the heart of this innovation is adaptive line-excitation. Rather than scanning a point of light, our method employs a short line of light that selectively illuminates only the regions of interest (ROIs), specifically the active neuronal cell bodies. This approach offers two key advantages over traditional methods. First, it samples a larger area of neurons simultaneously, which accelerates the imaging process by 5 to 10 times, depending on the line shape. Second, it targets only the active regions and avoids imaging the background areas, so the total laser power delivered to the brain tissue can be reduced by more than tenfold, depending on the labeling density. This significantly lowers the risk of photodamage.
The adaptive line-excitation method uses a digital micromirror device (DMD) with thousands of tiny mirrors to dynamically shape and steer the light beam. The laser beam is first shaped into a short line and directed to the DMD, which encodes the ROIs’ morphologies. The diffracted beam from the DMD carries the ROI information and illuminates the corresponding sample area. This approach precisely targets active neurons and avoids unnecessary illumination, which enhances imaging safety and efficiency.
We demonstrated the effectiveness of this new microscope by conducting high-speed calcium imaging of neuronal activity in the mouse cortex. The system successfully captured rapid neuronal events in real time at frame rates of up to 198 Hz, significantly surpassing the capabilities of conventional two-photon microscopes. Depending on the occupancy of neuronal areas within the field of view, the effective illumination power after the DMD can be reduced by more than tenfold compared with conventional light-delivery methods.
In our view, the development of high-speed adaptive line-excitation microscopy represents a significant advancement in neural imaging, offering a faster, safer and more efficient means to study the brain. This innovation not only expands the capabilities of two-photon microscopy but also opens new avenues for research in neuroscience and other fields requiring precise and dynamic imaging techniques.
Researchers
Yunyang Li, Shu Guo, Ben Mattison, Junjie Hu, Kwun Nok Mimi Man* and Weijian Yang, University of California, Davis, USA
*Present address: Max Planck Florida Institute for Neuroscience, USA
References
1. W. Denk et al. Science 248, 73 (1990).
2. W. Yang et al. Nat. Methods 14, 349 (2017).
3. Y. Li et al. Optica 11, 1138 (2024).