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Holey carbon grid

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Quantifoil holey carbon grids are thin film support structures used in electron microscopy. They consist of a perforated carbon film applied to a metal mesh. The holes in the carbon film allow for the imaging of specimens suspended in the open areas.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (16 mentions) K2 summit direct electron detector, by Ametek (5 mentions) Vitrobot, by Thermo Fisher Scientific (5 mentions) Titan krios, by Thermo Fisher Scientific (4 mentions) Whatman, by Cytiva (3 mentions) Mark 4, by Thermo Fisher Scientific (3 mentions) Polara microscope 300 kv, by Thermo Fisher Scientific (3 mentions) K2 summit ded camera, by Ametek (3 mentions) K2 summit direct detector, by Ametek (3 mentions) Titan krios, by Ametek (2 mentions) K2 summit, by Ametek (2 mentions) Ultrascan 4 4 k ccd, by Ametek (2 mentions) Titan krios electron microscope, by Thermo Fisher Scientific (2 mentions) 10 nm colloidal gold beads, by Merck Group (2 mentions) Pdc 002, by Harrick (2 mentions) K3 summit direct electron detector, by Ametek (2 mentions) Polara electron microscope, by Thermo Fisher Scientific (2 mentions) Titan krios g3i microscope, by Thermo Fisher Scientific (2 mentions) Trpa1 to 1.2 1 3 holey carbon grids, by Quantifoil (2 mentions) Polara and titan krios microscopes, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Glow-discharged holey carbon grids (16 citations) Quantifoil R1.2/1.3 300-mesh gold holey carbon grid (11 citations) Quantifoil R2/1 holey carbon grids (9 citations) Holey carbon-film grid (3 citations) Glow-discharged gold holey carbon 2/1 300-mesh grid (2 citations) Holey carbon TEM grid (2 citations) Quantifoil R2/1 holey carbon copper grid (2 citations) Glow-discharged R1.2/1.3 holey carbon grid (2 citations) Glow-discharged holey carbon R2/1 Cu 200 mesh grid (2 citations) R2/2 200 mesh holey carbon EM grid (2 citations)

59 protocols using holey carbon grid

1

Specimen Preparation for Cryo-EM

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Grids for transmission electron microscopy were prepared as described in Kowal et al.29 (link): 4 µl of the MloK1 2D crystal solution (0.8 mg/ml) were applied to glow-discharged Quantifoil holey carbon grids (R3.5/1, Cu 400 mesh), which had been coated with an additional <3-nm-thin amorphous carbon layer. Using an FEI Vitrobot IV with the environmental chamber set at 90% humidity and 20 °C, excess solution on the grid was blotted for 3.5 s, and the grids were then flash-frozen into liquid ethane.
Righetto R.D., Biyani N., Kowal J., Chami M, & Stahlberg H. (2019). Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM. Nature Communications, 10, 1722.
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2

Cryo-EM Imaging of Protein Samples

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Sample was placed on holey carbon grids (Quantifoil GmbH, Germany) and plunge-frozen into liquid ethane cooled down to LN2 temperature using a Vitrobot MK4 (FEI Corp, The Netherlands). Frozen grids were stored in LN2 and directly observed in a Titan Krios (FEI Corp, The Netherlands) operated at 300 kV and quipped with a K2 Summit direct electron detector (Gatan, Pleasanton, CA). All images were acquired in a single two-day session at a defocus range of 0.5 – 1.5 µm. Images were recorded in dose fractionation mode, with a dose rate 3–4 e−/pix/second, exposures per image sub-frames between 1 and 1.5 e−/ Å2 and a cumulative dose for the entire image series of 30 e−/ Å2. The final pixel size for the resulting 4k images was 1.0 Å/pix at the sample level.
Kudryashev M., Wang R.Y., Brackmann M., Scherer S., Maier T., Baker D., DiMaio F., Stahlberg H., Egelman E.H, & Basler M. (2015). Structure of the Type VI secretion system contractile sheath. Cell, 160(5), 952-962.
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3

Cryo-EM Imaging of SERT-Antibody Complexes

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The SERT-antibody complexes (2.5 µl) at a concentration of 40–80 µM were applied to glow-discharged Quantifoil holey carbon grids (gold, 1.2/1.3 or 2.0/2.0 µm size/hole space, 200 mesh). For ‘multi-shot’ data collection37 (link), 100 µM fluorinated n-octyl-β-d-maltoside (final concentration) was added to the sample prior to freezing. The grids were blotted for 1.5–2.5 s at 100% humidity using a Vitrobot Mark IV, followed by plunging into liquid ethane cooled by liquid nitrogen. Images were acquired using a FEI Titan Krios equipped with a Gatan Image Filter operating at 300 kV or an Arctica transmission electron microscope (TEM) at 200 kV. A Gatan K2 Summit direct electron detector was used, on both TEMs, to record movies in super-resolution counting mode with a binned pixel size of 1.044 or 0.823 Å/pixel on the Krios or 0.910 Å/pixel on the Arctica, respectively. The defocus values ranged from −1.0 to −2.5 µm. Exposures of 8–10 s were dose-fractionated into 40–100 frames, resulting in a total dose of 50–60 e−/Å2. Images were recorded using the automated acquisition program SerialEM37 (link).
Coleman J.A., Yang D., Zhao Z., Wen P.C., Yoshioka C., Tajkhorshid E, & Gouaux E. (2019). Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport. Nature, 569(7754), 141-145.
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4

Cryo-EM Sample Preparation Protocol

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The sample was frozen using a FEI Vitrobot Mark III. The sample was applied to Holey carbon grids (3.5/1, 400 mesh, Quantifoil) at 4 °C, 100% humidity and incubated on the grid for 60 s. A blot force of 10 and a blot time of 10–12 s was used before plunge freezing into liquid ethane. Samples were stored in liquid nitrogen temperatures before imaging.
Peng R., Rochon K., Stagg S.M, & Mears J.A. (2024). The Structure of the Drp1 Lattice on Membrane. bioRxiv.
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5

Isolation and Cryo-EM Analysis of Minicells

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To isolate minicells, overnight broth cultures (containing 0.2% arabinose for TH16943 or 100 μg/ml spectinomycin for maintenance of pBS58 in MS1868 and IJ1043) were centrifuged at 12,000 rpm for 10min to remove large cells. Supernatants were then centrifuged at 23,000 rpm for 20min, and the minicell pellet was resuspended in broth containing 100 μg/ml chloramphenicol to prevent phage gene expression. Minicells were infected with SP6 at a multiplicity of 2–10 and incubated at 37°C for up to 60 min. Portions of the infected cultures were mixed with 10 nm colloidal gold as fiducial markers, and 5 μl samples were applied to freshly glow-discharged holey carbon grids (Quantifoil Micro Tools, GmbH, Jena, Germany). After briefly blotting with filter paper to remove excess liquid, grids were rapidly frozen in liquid ethane using a gravity-driven plunger, and were stored in liquid nitrogen.
Tu J., Park T., Morado D., Hughes K.T., Molineux I.J, & Liu J. (2017). Dual host specificity of phage SP6 is facilitated by tailspike rotation. Virology, 507, 206-215.
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6

cVLP Morphology Studied by CryoTEM

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cVLP morphology was studied by CryoTEM. A 2–3 μL amount of sample was blotted onto holey carbon grids (Quantifoil Micro Tools, Großloebichau, Germany and Micro to Nano, Haarlem, Netherlands) previously glow discharged in a PELCO easiGlow glow discharger unit. The samples were subsequently plunged into liquid ethane at −180 °C using a Leica EM GP cryo workstation and observed in a JEM 2011 electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. During imaging, samples were maintained at −173 °C, and pictures were taken using a CCD-multiscan camera (Model No. 895, Gatan Inc., Pleasanton, CA, USA).
Fontana D., Garay E., Cervera L., Kratje R., Prieto C, & Gòdia F. (2021). Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus. Vaccines, 9(3), 251.
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7

CVD Graphene for C70 Thin Film Deposition

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Graphene was synthesized using chemical vapor deposition (CVD)40 (link). Twenty-five micrometer-thick copper foil was used as the synthesis substrate. CVD graphene was transferred to Quantifoil holey carbon grids via direct transfer18 (link). C70 films were deposited onto graphene TEM grids by thermal evaporation. Before the thermal evaporation process, TEM grids were pre-annealed in the air at 200 °C for 30 min to minimize surface adsorbate on graphene. The C70 films with thicknesses ranging from 0.5 to 10 nm were deposited at the deposition rate of 0.05 Å s−1 under a vacuum pressure of 2 × 10−6 Torr. The graphene substrate was held at 110 °C during the deposition.
Choe J., Lee Y., Park J., Kim Y., Kim C.U, & Kim K. (2019). Direct imaging of structural disordering and heterogeneous dynamics of fullerene molecular liquid. Nature Communications, 10, 4395.
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8

Cryo-EM Grid Preparation Protocol

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Four microliters of biotin eluate was applied to holey carbon grids (R2/1 Cu, 200 mesh, with or without 2-nm ultrathin carbon film; Quantifoil) freshly cleaned by glow discharge with negative polarity at 20 mA for 20 sec using an EMS GloQube (Quorum Technologies). Grids were plunge-frozen in a liquid ethane/propane mix using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4°C and 95% humidity.
Bonneau F., Basquin J., Steigenberger B., Schäfer T., Schäfer I.B, & Conti E. (2023). Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA–RNA interactions. Genes & Development, 37(11-12), 505-517.
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9

Mitochondrial Cryo-EM Imaging Protocol

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For analysis by electron cryo-microscopy, mitochondria were washed twice with 320 mM trehalose, 20 mM Tris, pH 7.3, and 1 mM EGTA. Samples were mixed 1:1 with fiducial gold markers (10 nm gold particles conjugated to protein A; Aurion), blotted, and immediately plunge-frozen in liquid ethane on holey carbon grids (Quantifoil Micro Tools). Single tilt series (±60°, step size 1.5°) were collected on a Polara microscope (300 kV; FEI) using an Ultrascan 4 × 4k CCD (Gatan, Inc.) and a post-column Quantum energy filter (Gatan, Inc.) at −9 µm defocus. The nominal magnification was 34,000×, resulting in a pixel size of 6 Å. A total dose of ∼130 e−/Å2 was used. Tilt series alignment using the gold fiducial markers and tomogram reconstruction by back-projection were performed using the IMOD software package (Kremer et al., 1996 (link)). To increase contrast, a final filtering step applying nonlinear anisotropic diffusion (Frangakis and Hegerl, 2001 (link)) was performed. Manual segmentation was performed with the program Amira (FEI).
Mourier A., Motori E., Brandt T., Lagouge M., Atanassov I., Galinier A., Rappl G., Brodesser S., Hultenby K., Dieterich C, & Larsson N.G. (2015). Mitofusin 2 is required to maintain mitochondrial coenzyme Q levels. The Journal of Cell Biology, 208(4), 429-442.
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10

Cryo-EM Structure of YicC-RNA Complex

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YicC-RNA complex sample at a concentration of 5 mg/ml was supplemented with 0.05% DDM detergent immediately before plunge-freezing to enable even distribution of particles on the grid. Then 3 µl of sample was applied to glow-discharged Quantifoil holey carbon grids (Cu, R1.2/1.3, 300 mesh). The grids were blotted for 2 s and plunged into liquid ethane with a Vitrobot plunger (4°C and 90% humidity). Cryo-EM data were collected with a Titan Krios microscope (FEI) operated at 300 kV and images were collected at a nominal magnification of 81,000 corresponding to a pixel size of 1.07 Å with a defocus range of −0.5 to −2 µm. The images were recorded on K3 electron direct detector in super-resolution mode at the end of a GIF-Quantum energy filter operated with a slit width of 15 eV. For data collection with a K3 detector, a dose rate of 15 electrons per pixel per second and an exposure time of 3.82 s were used, generating 70 video frames with a total dose of 60 electrons per Å2. The statistics for the cryo-EM data are listed in Table 2.
Wu R., Barnes S., Dahlin H., Khamrui S., Ingle S., Xiang Y., Shi Y., Bechhofer D, & Lazarus M. (2023). Structural insights into RNA cleavage by a novel family of bacterial RNases. Research Square.
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