Cryo-electron tomography (cryo-ET) for in situ structure analysis is a key technique for studying the in situ high-resolution structure of biomacromolecular complexes and their interactions. However, limited by the penetration ability of the electron beam, it is necessary to use the focused ion beam (cryo-FIB) to thin the cell and tissue samples into thin slices of about 200 nanometers before collecting cryo-ET data. Cryo-photonic correlation imaging technology can provide fluorescence positioning guidance for the precise preparation of cryo-FIB frozen aqueous sections containing specific target structures, but the optical resolution of the cryo-fluorescence microscope and the alignment accuracy of the light and electron microscope images are the key factors that restrict the success rate of the cryo-electrocorrelation experiment.

In order to solve the above technical bottlenecks, the Bioimaging Center of the Protein Science Research Platform of the Institute of Biophysics, Chinese Academy of Sciences has been committed to the development of new cryo-optical correlation imaging technology. Based on the high-vacuum optical cold stage HOPE (Journal of Structural Biology, 2017), which was independently developed in the early stage, the cryo-structured light imaging system HOPE-SIM was successfully developed by introducing the structured light illumination imaging technology, realizing the horizontal Optical resolution better than 200 nanometers, and light mirror-focused ion beam three-dimensional correlation alignment accuracy better than 150 nanometers.

Correlative Light and Electron Microscopy (CLEM) uses fluorescent specific markers to trace specific biological macromolecules or subcellular structures to achieve three-dimensional fluorescence positioning imaging of the entire cell, and then through the registration of the fluorescence image and the electron microscope image, the correlation information of the fluorescent marker signal and the ultrastructure of the electron microscope is obtained.

One of the application directions of cryo-photocorrelation imaging technology is to indicate the specific position of the structure of the fluorescent label in the electron microscope image through the correlation image, and realize the electron microscope high-resolution structure analysis of the fluorescent traced target. Thanks to the non-destructive characteristics of light microscopy imaging on biological samples, the three-dimensional fluorescence positioning information inside the sample can be obtained without damaging the sample. Then, through the photoelectric correlative imaging process and correlative alignment software, the three-dimensional fluorescence image is correlatively matched with the scanning electron microscope image, and the thinning process of the target area by cryo-FIB is realized under the guidance of the fluorescent signal. In this way, “blind cutting” can be avoided, and the guided cutting of the fluorescent indicator target can be realized, so as to improve the efficiency of cryo-focused ion beam technology for electron tomography slice sample preparation.

At present, there are many types of optoelectronic correlation imaging to guide the cryo-FIB thinning technology process. According to the system configuration, it can be divided into optical microscope electron microscope split photoelectric correlation imaging system and integrated photoelectric correlation imaging system. Since 2013, the technical team of the Bioimaging Center has been focusing on the methodology research of cryo-electro-optical correlation imaging technology. In terms of the development of light microscopy and electron microscopy split-type photoelectric correlation imaging system, in 2017, it independently developed a high-vacuum optical cold stage HOPE (High-vacuum Optical Platform for cryo-CLEM) that can be mounted on an inverted fluorescence microscope. Vacuum optical cold stage HOPE (High-vacuum Optical Platform for cryo-CLEM). HOPE can be adapted and connected with the frozen sample rod of the transmission electron microscope. After the fluorescence positioning is completed, the sample will be transferred into the electron microscope with the frozen sample rod for high-resolution data acquisition. At the same time, combined with the photoelectric correlation positioning software, it can realize the matching of large field of view optical positioning imaging and electron microscope imaging. HOPE uses a frozen sample rod to realize cryo-light microscopy imaging, cryo-transmission and cryo-TEM imaging, which effectively avoids repeated clamping of the cryo-grid during photoelectric correlation imaging, ensures the integrity and identity of frozen samples, and effectively improves correlation success rate and experimental efficiency.

However, the HOPE system based on wide-field imaging technology is limited by the optical diffraction limit and the space limitation of the cryo-optical imaging device. It is 400-500 nanometers, and the longitudinal resolution reaches micron level, which is very unfavorable for accurately capturing the target structure of hundreds of nanometers in cells with a thickness of several microns.

Under the premise that the structured light illumination super-resolution fluorescence imaging technology can double the resolution of the wide-field fluorescence microscope, it also has technical advantages such as no need for special fluorescent probes, fast imaging speed, and low irradiance density. Among the techniques, it is the most suitable for high-resolution imaging of frozen samples in a frozen environment. Therefore, the research team chose structured light illumination imaging technology as a means to improve the resolution of cryo-fluorescence imaging, and independently developed a large chamber high-vacuum cold stage based on an inverted fluorescence microscope. The chamber has a built-in 0.9NA long working distance optical objective lens and an anti-pollution device system (ACS and cryo-box), external vacuum transfer system (TPS), and cryo-EM sample holder (cryo-holder) adapter. At the same time, with the help of the three-dimensional structured light illumination (SIM) optical path, three-dimensional structured light illumination imaging of frozen samples in a vacuum environment is realized. While improving the resolution of cryo-light microscopy, it effectively enhances the protection of frozen samples during photoelectric correlation imaging sample transfer.

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