Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

doi:10.1007/s40242-024-4006-8

高等学校化学研究 ›› 2024, Vol. 40 ›› Issue (6): 994-1003.doi: 10.1007/s40242-024-4006-8

Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

SHAO Lina¹, SUN Minglu^2,3, ZHANG Jinrui¹, XU Haijiao¹, YANG Hui^4,5, LI Hongru¹, WANG Huili¹, CUI Zongqiang⁴, LI Dayu^2,3, WANG Hongda^1,6,7

1. State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China;
2. State Key Laboratory of Applied Optics, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130022, P. R. China;
3. Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, P. R. China;
4. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, P. R. China;
5. University of Chinese Academy of Sciences, Beijing 100049, P. R. China;
6. School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230027, P. R. China;
7. Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266200, P. R. China

收稿日期:2024-01-08 出版日期:2024-12-01 发布日期:2024-10-26
通讯作者: WANG Hongda,hdwang@ciac.ac.cn;LI Dayu,lidayu@ciomp.ac.cn E-mail:hdwang@ciac.ac.cn;lidayu@ciomp.ac.cn
基金资助:
This work was supported by the Scientific Instrument Developing Project of the Chinese Academy of Sciences (No. ZDKYYQ20220005) and the National Natural Science Foundation of China (Nos. 22150003, and 22327808). Chinese patent applications have been submitted for relevant technologies (No. 202011538556.8, No. 202110126844.0 & No. ZL 202120587363.5).

Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

SHAO Lina¹, SUN Minglu^2,3, ZHANG Jinrui¹, XU Haijiao¹, YANG Hui^4,5, LI Hongru¹, WANG Huili¹, CUI Zongqiang⁴, LI Dayu^2,3, WANG Hongda^1,6,7

1. State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China;
2. State Key Laboratory of Applied Optics, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130022, P. R. China;
3. Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, P. R. China;
4. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, P. R. China;
5. University of Chinese Academy of Sciences, Beijing 100049, P. R. China;
6. School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230027, P. R. China;
7. Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266200, P. R. China

Received:2024-01-08 Online:2024-12-01 Published:2024-10-26
Contact: WANG Hongda,hdwang@ciac.ac.cn;LI Dayu,lidayu@ciomp.ac.cn E-mail:hdwang@ciac.ac.cn;lidayu@ciomp.ac.cn
Supported by:
This work was supported by the Scientific Instrument Developing Project of the Chinese Academy of Sciences (No. ZDKYYQ20220005) and the National Natural Science Foundation of China (Nos. 22150003, and 22327808). Chinese patent applications have been submitted for relevant technologies (No. 202011538556.8, No. 202110126844.0 & No. ZL 202120587363.5).

摘要/Abstract

摘要： The ideal method for imaging living cells is one that allows for long-term, label-free observation in real-time with super-resolution capabilities. Such a method would overcome the drawbacks of phototoxicity and photobleaching associated with fluorescence labeling microscopy. Fourier ptychography is a promising label-free imaging technique that surpasses the diffraction limit of conventional microscopy while avoiding issues related to fluorescent labeling. However, previous Fourier ptychography microscopy (FPM) systems lacked high spatiotemporal resolution, preventing real-time observation of subcellular organelle structures in living cells. To address this limitation, we have developed a high-speed super-resolution microscope based hemispherical digital illumination (HDI) device. This system utilizes 61 light-emitting diodes (LEDs) to provide high-angle illumination with a numerical aperture (NA) of 0.98. By employing a 40×/0.6 NA objective lens, we have achieved lateral resolutions of around 150 nm, enabling us to capture images at a speed of over 1 Hz with the field of view measuring 118×118 μm². HDI-FPM allows for the monitoring of various cellular processes, such as vesicular transportation, mitochondrial fusion and division, as well as cell-cell fusion over a duration of 4 h. The development of HDI-FPM represents a significant advancement in label-free imaging of living cells. It offers a comprehensive understanding of the mechanisms underlying cellular activities.

关键词: Label-free, Super-resolution, Fourier ptychography microscopy, Long-term, Cellular organelle, SARS-CoV-2 virus-like particle

Abstract: The ideal method for imaging living cells is one that allows for long-term, label-free observation in real-time with super-resolution capabilities. Such a method would overcome the drawbacks of phototoxicity and photobleaching associated with fluorescence labeling microscopy. Fourier ptychography is a promising label-free imaging technique that surpasses the diffraction limit of conventional microscopy while avoiding issues related to fluorescent labeling. However, previous Fourier ptychography microscopy (FPM) systems lacked high spatiotemporal resolution, preventing real-time observation of subcellular organelle structures in living cells. To address this limitation, we have developed a high-speed super-resolution microscope based hemispherical digital illumination (HDI) device. This system utilizes 61 light-emitting diodes (LEDs) to provide high-angle illumination with a numerical aperture (NA) of 0.98. By employing a 40×/0.6 NA objective lens, we have achieved lateral resolutions of around 150 nm, enabling us to capture images at a speed of over 1 Hz with the field of view measuring 118×118 μm². HDI-FPM allows for the monitoring of various cellular processes, such as vesicular transportation, mitochondrial fusion and division, as well as cell-cell fusion over a duration of 4 h. The development of HDI-FPM represents a significant advancement in label-free imaging of living cells. It offers a comprehensive understanding of the mechanisms underlying cellular activities.

Key words: Label-free, Super-resolution, Fourier ptychography microscopy, Long-term, Cellular organelle, SARS-CoV-2 virus-like particle

SHAO Lina, SUN Minglu, ZHANG Jinrui, XU Haijiao, YANG Hui, LI Hongru, WANG Huili, CUI Zongqiang, LI Dayu, WANG Hongda. Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles[J]. 高等学校化学研究, 2024, 40(6): 994-1003.

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Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

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