High-vacuum optical platform for cryo-CLEM (HOPE):A new solution for non-integrated multiscale correlative light and electron microscopy
| Autor: | Shuoguo Li, Shengliu Wang, Yang Shi, Xu Wei, Lasse Hyldgaard Klausen, Gang Ji, Mingdong Dong, Xiaojun Huang, Wei Ding, Fei Sun, Xiang Zhang, Tongxin Niu |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Materials science Vacuum Cryo-electron microscopy Correlative light and electron microscopy viruses CRYOELECTRON TOMOGRAPHY Green Fluorescent Proteins Ultra-high vacuum 02 engineering and technology macromolecular substances environment and public health law.invention Rats Sprague-Dawley 03 medical and health sciences Optics Structural Biology law Multifocal plane microscopy Microscopy Escherichia coli Animals FLUORESCENCE Cells Cultured Cryo-fluorescence microscopy Neurons business.industry Adapter (computing) Cryoelectron Microscopy Temperature Reproducibility of Results Wide field fluorescence imaging 021001 nanoscience & nanotechnology High-vacuum optical platform Dark field microscopy diagnosis Lens (optics) Luminescent Proteins PRECISION ALIGNMENT 030104 developmental biology Animals Newborn Microscopy Fluorescence CELLS Electron microscope 0210 nano-technology business Algorithms |
| Zdroj: | Li, S, Ji, G, Shi, Y, Klausen, L H, Niu, T, Wang, S, Huang, X, Ding, W, Zhang, X, Dong, M, Xu, W & Sun, F 2018, ' High-vacuum optical platform for cryo-CLEM (HOPE) : A new solution for non-integrated multiscale correlative light and electron microscopy ', Journal of Structural Biology, vol. 201, no. 1, pp. 63-75 . https://doi.org/10.1016/j.jsb.2017.11.002 |
| DOI: | 10.1016/j.jsb.2017.11.002 |
| Popis: | Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique thigh-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The 'touch-free' specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope. |
| Databáze: | OpenAIRE |
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