CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

Yang, Jie; Larson, Matthew R.; Sibert, Bryan; Shrum, Samantha; Wright, Elizabeth

doi:10.1016/j.jsb.2021.107709

Cited by 35 publications

(25 citation statements)

References 122 publications

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“…In our study, these options were valuable in instances where the timing of cell preparation and seeding is critical such as for primary Drosophila neurons and RSV-infection of BEAS-2B cells. We prepare grids in a general biosafety-level (BSL-2) lab setting using clean tools, sterile solutions, and include antibiotics/antimycotics in the growth media 6, [19][20][21] . For samples particularly sensitive to microbial contamination, the anti-fouling layer and ECM can be applied in a tissue culture hood or other sterile environment.…”

Section: Resultsmentioning

confidence: 99%

“…We prepare grids in a general biosafety-level 2 (BSL-2) lab setting using clean tools, sterile solutions, and include antibiotics/antimycotics in the growth media 6,19-21 . For samples particularly sensitive to microbial contamination, the anti-fouling layer and ECM can be applied in a tissue culture hood or other sterile environment.…”

Section: Resultsmentioning

confidence: 99%

“…The placement and positioning of cells can be used as fiducial markers for correlation in cryo-CLEM experiments, reducing the need for fragile finder-grids and fluorescent fiducial markers. However, it should be noted that such fiducial markers may still be useful for sub-micron accuracy correlation 19,33 . Furthermore, an even distribution of isolated cells is also highly beneficial for FIB-SEM experiments to maximize the number of cells from which lamella can be cut 15 .…”

Section: Discussionmentioning

confidence: 99%

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Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

et al. 2021

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Whole-cell cryo-electron tomography (cryo-ET) is a powerful technique that can provide nanometer-level resolution of biological structures within the cellular context and in a near-native frozen-hydrated state. It remains a challenge to culture or adhere cells on TEM grids in a manner that is suitable for tomography while preserving the physiological state of the cells. Here, we demonstrate the versatility of micropatterning to direct and promote growth of both cultured and primary eukaryotic cells on TEM grids. We show that micropatterning is compatible with and can be used to enhance studies of host-pathogen interactions using respiratory syncytial virus infected BEAS-2B cells as an example. We demonstrate the ability to use whole-cell tomography of primary Drosophila neuronal cells to identify organelles and cytoskeletal stuctures in cellular axons and the potential for micropatterning to dramatically increase throughput for these studies. During micropatterning, cell growth is targeted by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design make micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (FIB-SEM).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

et al. 2021

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“…distances to landmarks), the registration and map tools available within SerialEM 20 , or external software such as CorRelator 21 .…”

Section: Transfer Positions Either Manually (By Measuringmentioning

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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“…In order to study the native structure of RSV and other enveloped viruses, we and others use whole-cell cryo-ET [19, 29, 43-45]. Cells are grown and infected with RSV directly on TEM grids [6, 46, 47]. The grids are then plunge-frozen to rapidly cryo-preserve the cells, associated viruses, and released virions in vitreous ice.…”

Section: Introductionmentioning

confidence: 99%

Respiratory syncytial virus matrix protein assembles as a lattice with local and extended order that coordinates the position of the fusion glycoprotein

Sibert

Kim

Yang

et al. 2021

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Respiratory syncytial virus (RSV) is a significant cause of respiratory illness in young children and adults worldwide. There is currently no vaccine or targeted antiviral for RSV. RSV is an enveloped, filamentous, negative-strand RNA virus. Individual virions vary in both diameter and length, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 μm in length. The RSV matrix (M) protein is peripherally associated with the interior of the viral membrane. Though the general arrangement of structural proteins within the virion is known, the molecular organization of M and other structural proteins was previously unknown. Here, using whole-cell cryo-electron tomography and sub-tomogram averaging, we show that M is arranged in a packed helical-like lattice of M-dimers ordered at an angle of ~47˚ to the viral long axis. Sub-tomogram averages including F and M indicate that the position of F on the viral surface is correlated with the underlying M lattice. Finally, we report that RSV F is frequently observed as pairs, with the F trimers oriented in an anti-parallel conformation to support potential interaction between trimers. These results provide insight into RSV assembly and virion organization and may aid in the identification and development of RSV vaccines and anti-viral targets.

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CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

Cited by 35 publications

References 122 publications

Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

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

Respiratory syncytial virus matrix protein assembles as a lattice with local and extended order that coordinates the position of the fusion glycoprotein

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