Lattice Micropatterning of Electron Microscopy Grids for Improved Cellular Cryo-Electron Tomography Throughput

Engel, Leeya; Vasquez, Claudia G.; Montabana, Elizabeth; Sow, Belle M.; Walkiewicz, Marcin P.; Weis, William I.; Dunn, Alexander R.

doi:10.1016/j.bpj.2020.11.1218

Cited by 5 publications

(12 citation statements)

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“…One significant limiting factor for whole-cell cryo-ET workflows has been obtaining sufficient numbers of collectable targets per grid. Recently, a number of groups have developed protocols for micropatterning grids for cryo-EM, with one advantage being improved data collection efficiency [15][16][17] . Here we use micropatterning to optimize cryo-ET studies of primary Drosophila neurons and cultured human cell lines (uninfected or RSV-infected).…”

Section: Discussionmentioning

confidence: 99%

“…A single seeding step at an optimized cell density, as described in the methods for Drosophila neurons, is a viable option for most cell types. However, it is also possible to seed cells onto the substrate at a lower initial concentration and the add more cells in a monitored fashion as described here and in other literature 17 . This sequential seeding can provide more consistent results in some cases.…”

Section: Discussionmentioning

confidence: 99%

“…Bringing micropatterning to cryo-ET will not only increase throughput, but it can also open up new studies for exploring complex and dynamic cellular microenvironments. Recently, we and others have begun using micropatterning techniques on TEM grids through multiple approaches [15][16][17] . Here, we describe the use of a maskless photopatterning technique for TEM grids using the Alvéole PRIMO system.…”

Section: Introductionmentioning

confidence: 99%

“…ECM proteins can then be added to the patterns for the appropriate cell culture. This method has been used by several groups for cryo-ET studies of RPE1, MDCKII, HFF, and endothelial cell lines [15][16][17] . The PRIMO system is compatible with multiple anti-fouling layer substrates as well as either a liquid or gel photocatalyst reagent.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we and others have begun using micropatterning techniques on TEM grids through multiple approaches 15-17 . Here, we describe the use of a maskless photopatterning technique for TEM grids using the Alvéole PRIMO system.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

Preprint

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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: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2021

Preprint

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Technological improvements for whole cell cryo-ET of respiratory syncytial virus infected cells

et al. 2021

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Respiratory syncytial virus (RSV) is a common respiratory virus that causes over 235,000 hospitalizations in the US each year [1,2]. There are currently no approved treatments or vaccines. RSV virions are enveloped filamentous virions that are highly pleomorphic, but generally range in length from 1-4 µm with a diameter of ~130 nm [3]. The RSV matrix protein (M) has been crystallized as both a monomer and a dimer and experiments have shown that dimerization is required to assemble filamentous virions [4,5]. However, the organization of matrix within the virion has not been determined. The structure and organization matrix proteins or matrix protein equivalents of other paramyxoviruses has been solved by cryo-EM, cryo-ET, and sub-tomogram averaging [6][7][8].

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Waffle Method: A general and flexible approach for improving throughput in FIB-milling

et al. 2022

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Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the ‘Waffle Method’ which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

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Lattice Micropatterning of Electron Microscopy Grids for Improved Cellular Cryo-Electron Tomography Throughput

Cited by 5 publications

References 0 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

Technological improvements for whole cell cryo-ET of respiratory syncytial virus infected cells

Waffle Method: A general and flexible approach for improving throughput in FIB-milling

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