Cryogenic superresolution correlative light and electron microscopy on the frontier of subcellular imaging

Niu

et al. 2023

Commun Biol

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

Section: Introductionmentioning

confidence: 99%

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Niu

et al. 2023

Commun Biol

“…Then, the fluorescence image can be used to navigate cryo-FIB fabrication. The conventional cryo-CLEM workflow requires a stand-alone fluorescence microscope with a cryostage for cryo-fluorescence imaging 11,12,14,27,28 , followed by cryo-scanning electron microscopy (cryo-SEM). A correlation alignment between the cryo-FM and cryo-SEM images is generated and used to guide cryo-FIB milling 15,[29][30][31] .…”

Section: Introductionmentioning

confidence: 99%

ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study

Wang

et al. 2022

Preprint

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent in situ cryo-ET structural study. Here, we developed a precise cryogenic fabrication system, the ELI trifocal microscope (ELI-TriScope), by setting up an electron (E) beam, a light (L) beam and an ion (I) beam at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella without sacrificing the throughput. ELI-TriScope was developed starting from a commercial dual-beam scanning electron microscope (SEM) by incorporating a cryo-holder-based transfer system and embedding an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam (FIB) milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome, with an ~100% success rate, and discovered new in situ structural features of the human centrosome through a subsequent cryo-ET structural study.

“…1) 9,[16][17][18][19][20] . This workflow normally requires a stand-alone fluorescence microscope with a cryo-stage for cryo-fluorescence imaging [12][13][14]21,22 , followed by cryo-scanning electron microscopy (cryo-SEM). A correlation alignment between the cryo-FM and cryo-SEM images is generated and used to guide cryo-FIB milling 9,15,18,23 .…”

Section: Introductionmentioning

confidence: 99%

“…The correlation accuracy and success rate between cryo-FM and cryo-FIB is limited by the resolution of cryo-FM and further attenuated by the factors of specimen deformation, devitrification, and ice contamination 7,21 . We previously developed a unique high-vacuum optical platform for cryo-CLEM (HOPE) 22 , which utilized an integrative cryo-holder and a specific vacuum chamber to minimize ice contamination and reduce the risk of specimen deformation and devitrification during cryo-FM imaging and specimen transfer.…”

Section: Introductionmentioning

confidence: 99%

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Niu

et al. 2022

Preprint

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we developed a new cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 150 nm enough for the subsequent cryo-lamella fabrication. We successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.