Tecnai arctica microscope

Manufactured by Thermo Fisher Scientific

Sourced in Netherlands

The Tecnai Arctica is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of biological samples. It features a stable, high-brightness electron source and advanced optics to provide excellent image quality and resolution. The Tecnai Arctica is capable of resolving features at the nanometer scale, making it a valuable tool for structural biology and materials science research.

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14 protocols using tecnai arctica microscope

Cryo-EM Imaging of Human PANX1 Protein

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The cryo-EM grids were prepared using Vitrobot Mark IV (FEI) operated at 4 °C and 100% humidity. For samples of hPANX1 in digitonin, 4 μl aliquots of samples at concentrations of approximately 8 mg/ml were applied onto glow-discharged holey carbon grids (Quantifoil R1.2/1.3) 300 mesh Au grid. After a waiting time of 5 s, the grids were blotted for 2 s and plunged into liquid ethane for quick freezing.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). The qualified grids were transferred into a Titan Krios microscope (FEI) operated at 300 kV for data acquisition equipped with Gatan K2 Summit detector and GIF Quantum energy filter. Images were automatically recorded using AutoEMation with a slit width of 20 eV on the energy filter and in the super-resolution mode at a nominal magnification of 130,000×, corresponding to a calibrated pixel size of 1.08 Å at the object scale, and with defocus ranging from 1.4 to 1.9 μm. Each stack was exposed for 5.6 s with an exposing time of 0.175 s per frame, resulting in a total of 32 frames per stack, and the total dose rate for each stack was about 50 e/Å².

Zhang S., Yuan B., Lam J.H., Zhou J., Zhou X., Ramos-Mandujano G., Tian X., Liu Y., Han R., Li Y., Gao X., Li M, & Yang M. (2021). Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis. Cell Discovery, 7, 30.

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Cryo-EM Grid Preparation for CLC-7/Ostm1

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The cryo-EM grids were prepared using Vitrobot Mark IV (FEI) operated at 4°C and 100% humidity. For samples of CLC-7/Ostm1, 4-μl aliquots of samples at concentrations of approximately 8 mg/ml were applied onto glow-discharged holey carbon grids (Quantifoil R1.2/1.3) 300 mesh Au grid. After a waiting time of 5 s, the grids were blotted for 2 s and plunged into liquid ethane for quick freezing.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). The qualified grids were transferred into a Titan Krios microscope (FEI) operated at 300 kV for data acquisition, and the Gatan K2 Summit detector was equipped with a GIF Quantum energy filter. Images were automatically recorded using SerialEM with a slit width of 20 eV on the energy filter and in super-resolution mode at a nominal magnification of ×130,000, corresponding to a calibrated pixel size of 1.08 Å at object scale, and with defocus ranging from 1.3 to 2.3 μm. Each stack was exposed for 5.6 s with an exposing time of 0.175 s per frame, resulting in a total of 32 frames per stack, and the total dose rate for each stack was about 50 e/Å².

Zhang S., Liu Y., Zhang B., Zhou J., Li T., Liu Z., Li Y, & Yang M. (2020). Molecular insights into the human CLC-7/Ostm1 transporter. Science Advances, 6(33), eabb4747.

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Cryo-ET of S. aureus Cell Wall

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A volume of 3.5 μl from HF treated S. aureus sacculi mixed with BSA treated 10 nm gold was applied to Lacey carbon grids twice and double blotted before plunge freezing in liquid ethane using a Leica EM GP2 plunge freezer. Tomograms were collected on a FEI Tecnai Arctica microscope operated at 200 kV, with a Falcon III direct electron detector and Volta phase plate. Each tomogram was collected at a nominal defocus of 3 μm, with a total dose applied to the sample of 120 e/Å over ±60° in 2° increments, with a nominal pixel size of 4.3 Å. Tomograms were reconstructed with IMOD³⁷, using weighted back projection and a final binning of 3. Tomograms were filtered with nonlinear anisotropic diffusion (NAD) in order to improve contrast.

Pasquina-Lemonche L., Burns J., Turner R.D., Kumar S., Tank R., Mullin N., Wilson J.S., Chakrabarti B., Bullough P.A., Foster S.J, & Hobbs J.K. (2020). The Architecture of the Gram Positive Bacterial Cell Wall. Nature, 582(7811), 294-297.

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Cryo-TEM of Pickering Nanoemulsions

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Imaging was performed using
an FEI Tecnai Arctica microscope operating
at an acceleration voltage of 200 kV. Cryo-TEM samples were prepared
by depositing 5 μL of a 0.5% w/w dodecane-in-water or squalane-in-water
Pickering nanoemulsion onto a plasma-treated Quantifoil holey carbon-coated
copper grid, followed by blotting for approximately 4 s and then plunging
into a pool of liquid ethane to vitrify the sample using a Leica EM
GP automatic plunge freezer (25 °C, 99% humidity). Transfer of
the vitrified grids into a precooled cryo-TEM holder was performed
at −196 °C prior to microscopic analysis.

Hunter S.J., Chohan P., Varlas S, & Armes S.P. (2024). Effect of Temperature, Oil Type, and Copolymer Concentration on the Long-Term Stability of Oil-in-Water Pickering Nanoemulsions Prepared Using Diblock Copolymer Nanoparticles. Langmuir, 40(7), 3702-3714.

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Cryo-EM Data Collection with Gatan K2

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A 2.5-μl drop of 3 mg/ml RP solution was applied to a glow-discharged C-flat grid (R 1/1, 400 Mesh, Protochips, CA, USA), blotted for 2 s at 4 °C and 100% humidity, then plunged into liquid ethane and flash frozen using the FEI Vitrobot Mark IV. The cryo-grid was imaged in an FEI Tecnai Arctica microscope, equipped with an Autoloader, at a nominal magnification of 54,000 times and an acceleration voltage of 200 kV. Coma-free alignment was manually conducted prior to data collection. Cryo-EM data were collected semi-automatically by the Leginon version 3.1 (Suloway et al., 2005 (link)) on the Gatan K2 Summit direct detector camera (Gatan Inc., CA, USA) in a counting mode, with 9.0 s of total exposure time and 250 ms per frame. This resulted in movies of 36 frames per exposure and an accumulated dose of 50 electrons/Å². The calibrated physical pixel size is 0.98 Å. The defocus in data collection was set in the range of from -1.0 to -3.0 μm. A total of 20,000 movies were collected, among which 16,111 movies were selected for further data analysis.

Lu Y., Wu J., Dong Y., Chen S., Sun S., Ma Y.B., Ouyang Q., Finley D., Kirschner M.W, & Mao Y. (2017). Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle. Molecular cell, 67(2), 322-333.e6.

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Cryo-EM Sample Preparation and Imaging

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The cryo-EM grids were prepared using a Vitrobot Mark IV (FEI) operated at 8 °C and 100% humidity. For samples of hNKCC1 and mKCC2, 4 μl aliquots of fresh samples at concentrations of ~10 mg/ml were applied onto glow-discharged holey carbon grids, 300 mesh gold (Quantifoil R1.2/1.3). After 5 s, the grids were blotted for 2 s and plunged into liquid ethane for quick freezing.
The cryo-EM grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k × 4k camera (FEI). Qualified grids were transferred to a Titan Krios microscope (FEI) operated at 300 kV for data acquisition and equipped with Cs-corrector (ThermoFisher Scientific Inc.), Gatan K2 Summit detector, and GIF Quantum energy filter. Images were automatically recorded using AutoEMation with a slit width of 20 eV for the energy filter and in super-resolution mode at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 1.091 Å at object scale, and with defocus ranging from 1.4 to 1.9 μm. Each stack was exposed for a total of 5.6 s with an exposure of 0.175 s for each of 32 frames—the total dose for each stack was about 50 e⁻/Å².

Zhang S., Zhou J., Zhang Y., Liu T., Friedel P., Zhuo W., Somasekharan S., Roy K., Zhang L., Liu Y., Meng X., Deng H., Zeng W., Li G., Forbush B, & Yang M. (2021). The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2. Communications Biology, 4, 226.

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Cryo-EM Imaging of mTRPML1 Complexes

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The cryo-EM grids were prepared using Vitrobot Mark IV (FEI) at 4°C and 100% humidity. For samples of both mTRPML1 amphipols and nanodiscs, 4 μL aliquots of samples at concentrations of 0.5–0.7 mg/mL were applied onto glow-discharged holey carbon grids (Quantifoil R1.2/1.3). After a waiting time of 3 s, the grids were blotted for 1–2 s and plunged into liquid ethane for quick freezing.
The grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II direct electron detector (FEI). The qualified grids were transferred into a Titan Krios microscope (FEI) operated at 300 kV for data acquisition. Images were recorded using a K2 submit direct electron detector (Gatan) in a counting mode at a nominal magnification of 22,500×, corresponding to a calibrated pixel size of 1.32 Å at object scale, and with defocus ranging from 1.7–2.6 μm. Date acquisition were performed semi-automatically using UCSF-Image4 (Li et al., 2015 (link)) in a movie mode, with a dose rate of 8.2 counts (10.9 electrons) per pixel size per second for a total exposure time of 8 s. Each micrographs were stored as a movie stack with 32 frames.

Zhang S., Li N., Zeng W., Gao N, & Yang M. (2017). Cryo-EM structures of the mammalian endo-lysosomal TRPML1 channel elucidate the combined regulation mechanism. Protein & Cell, 8(11), 834-847.

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Cryo-ET of S. aureus Cell Wall

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Cryo-EM Imaging of Biomolecular Complexes

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Cryo-grids were screened on a Tecnai F20 (FEI) operated at a voltage of 200 kV with an eagle 4k x 4k CCD camera (FEI) or a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II 4k x 4k camera (FEI). The screened grids were transferred into a Titan Krios (FEI) operated at 300 kV and images were collected using Falcon II camera with a nominal magnification of 75000x, corresponding to a calibrated pixel size of 1.08 A ˚at object scale, and with defocus ranging from À1.5 mm to À3.0 mm. Data acquisition were performed automatically using AutoEMation2 (developed by J. Lei) in the movie mode with a dose rate of 25.5 e À A ˚À2 s À1 and a total exposure time of 1.6 s, which yields a stack of 26 frames for each micrograph.

Li N., Wu J.X., Ding D., Cheng J., Gao N, & Chen L. (2017). Structure of a Pancreatic ATP-Sensitive Potassium Channel. Cell, 168(1-2).

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Time-resolved Cryo-EM of Molecular Complexes

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The cryo-grids were initially screened in a 200 kV Tecnai Arctica microscope (Thermo Fisher). Good-quality grids were then transferred to a 300 kV Titan Krios G2 microscope (Thermo Fisher) equipped with the post-column BioQuantum energy filter (Gatan) connected to a K2 Summit direct electron detector (Gatan). Coma-free alignment and parallel illumination were manually optimized prior to each data collection session. Cryo-EM data were acquired automatically using SerialEM software^{45 (link)} in a super-resolution counting mode with 20 eV energy slit, with the nominal defocus set in the range of −0.8 to −2.0 μm. A total exposure time of 10 s with 250 ms per frame resulted in a 40-frame movie per exposure with an accumulated dose of ~50 electrons per Å². The calibrated physical pixel size and the super-resolution pixel size were 1.37 Å and 0.685 Å, respectively. For time-resolved sample conditions, 1,781, 2,298, 15,841, 2,073 and 2,071 movies were collected for cryo-grids made with the reaction time of 0, 0.5, 1, 5, and 10 min, respectively. For the condition of exchanging ATP to ATPγS at 1 min after substrate addition, 21,129 movies were collected.

Zhang S., Zou S., Yin D., Zhao L., Finley D., Wu Z, & Mao Y. (2022). USP14-regulated allostery of the human proteasome by time-resolved cryo-EM. Nature, 605(7910), 567-574.

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