Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells

Klein, Steffen; Wachsmuth-Melm, Moritz; Winter, Sophie L.; Kolovou, Androniki; Chlanda, Petr

doi:10.1016/bs.mcb.2020.12.009

Cited by 20 publications

(17 citation statements)

References 25 publications

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“…However, it has not yet been conclusively determined whether (a) mechanical stability or (b) matching thermal expansion coefficients (substrate vs. film) to reduce cryowrinkling 27 , is the most crucial factor for successful 3D correlation. Moreover, for picking up fragile Au grids, polydimethylsiloxane-coated dishes may be used 5 .…”

Section: Representative Resultsmentioning

confidence: 99%

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Bieber

Capitanio

Wilfling

et al. 2021

JoVE

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Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozenhydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

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Section: Representative Resultsmentioning

confidence: 99%

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Bieber

Capitanio

Wilfling

et al. 2021

JoVE

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“…In situ cryo-CLEM was performed on cryo-FIB milled samples based on a previously established method (Klein et al, 2021a(Klein et al, , 2021b, as described below:…”

Section: In Situ Cryo-correlative Light and Electron Microscopy (Cryo...mentioning

confidence: 99%

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion

Klein

Golani

Lolicato

et al. 2022

Preprint

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Interferon-induced transmembrane protein 3 (IFITM3) is a broad-spectrum restriction factor of viral entry, yet the molecular mechanism is poorly understood. We found that IFITM3 localizes to late endosomes, preventing viral fusion. Atomistic molecular dynamics simulations and continuum membrane modeling predicted IFITM3-induced local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier of fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography verified this model by visualizing influenza A virus-induced arrested membrane fusion at molecular resolution. Accumulated hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as the molecular mechanism of IFITM3. The presence of hemagglutinin in post-fusion conformation close to hemifusion sites further showed that IFITM3 does not interfere with the viral fusion machinery, which corroborates IFITM3-induced lipid sorting as the primary cause of hemifusion stabilization.

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“…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] . This workflow is complicated, and ensuring the sample quality is challenging due to multiple transfers between microscopes 30,32 .…”

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

Jia

et al. 2022

Preprint

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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.

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Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells

Cited by 20 publications

References 25 publications

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion

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

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