活细胞 z-bio 2025-01-13 830

　　Supported by the National Natural Science Foundation of China (Grant No. 32150015) and other grants, the team led by Zhang Yongdeng from the School of Life Sciences at Xihu University, in collaboration with Huang Xiaoshuai's team from Peking University, Fan Junchao's team from Chongqing University of Posts and Telecommunications, and Chen Liangyi's team from Peking University, has achieved breakthroughs in the development of rapid 3D super-resolution imaging technology for live cells. The related achievement, titled "Elucidating Subcellular Architecture and Dynamics at Isotropic 100 nm Resolution with 4Pi SIM Using 4Pi SIM Super Resolution Microscopic Imaging System," was published online on December 23, 2024 in the journal Nature Methods. Paper link: https://www.nature.com/articles/s41592-024-02515-z .

　　As the basic unit of life, cells have numerous organelles with different fine structures and division of labor, performing different life activities. Therefore, observing and studying the dynamic behavior of subcellular structures of living cells in three-dimensional space is of great significance for the development of cell biology. The 3D structured light illumination microscope (3D-SIM) can increase the 3D spatial resolution to twice that of a wide field microscope, and has the advantages of low phototoxicity, fast imaging speed, and compatibility with traditional fluorescent probes, making it particularly suitable for 3D delayed imaging of living cells. However, the axial resolution of 3D-SIM (about 300 nanometers) is much lower than the lateral resolution (about 100 nanometers), and this anisotropic resolution can lead to blurry image details and overall distortion.

　　In this project, based on the research experience of 4Pi single-molecule super-resolution microscope and Heisenberg structured light illumination microscope, the researchers carefully designed the optical and mechanical structures of 4Pi SIM microscope (as shown in the figure), minimizing the influence of thermal fluctuations and mechanical vibrations, and ensuring the long-term stability of six beam interference alignment and fluorescence interference; By introducing a focus locking module, the system can ensure precise alignment of two objective lenses; By designing an optical path difference adjustment module, the system can quickly and accurately fine tune the optical path difference between the upper and lower interference arms to zero; In addition, the system adopts a novel lighting module and improves the reconstruction algorithm to adaptively estimate and compensate for phase errors caused by mismatched optical path differences in the original data, thereby minimizing reconstruction artifacts during long-term imaging. These technological innovations enable the 4Pi SIM system to reveal various subcellular structures, including microtubules, endoplasmic reticulum, mitochondria, Golgi, etc., with extremely high clarity and detail.

　　In addition, 4Pi SIM has achieved three-dimensional isotropic 100 nanometer dynamic imaging on living cells for the first time, with an imaging time of several hours, capable of capturing hundreds of time points, and a maximum imaging speed of 0.7 Hz. Finally, 4Pi SIM also has the capability of simultaneous dual color imaging, which can capture the rapid interaction process between different organelles in three-dimensional space. In summary, 4Pi SIM demonstrates the true high fidelity three-dimensional isotropic resolution super-resolution imaging capability of live cells, taking an important step in the field of live cell super-resolution imaging and having great potential in elucidating subcellular dynamic behavior at the nanoscale.