Vitrobot system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Vitrobot system is a laboratory instrument designed for the preparation of samples for cryo-electron microscopy (cryo-EM) analysis. The core function of the Vitrobot system is to rapidly freeze hydrated samples, creating a thin vitreous ice layer that preserves the native structure of the biological molecules or complexes under investigation.

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Lab products found in correlation

18 protocols using vitrobot system

Nanoparticle Characterization by DLS and TEM

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DLS was done on a Malvern
Zetasizer Nano
S equipped with a laser operating at 633 nm. Sample grids for electron
microscopy were obtained from Electron Microscopy Sciences (EMS, Hatfield,
PA, USA) and were rendered hydrophilic using a plasma cleaning setup
(15 s at 10^–1 Torr). For cryoTEM, samples were cast
on Quantifoil R2/2 grids or 400 mesh holey carbon grids. After blotting,
samples were plunged into liquid ethane using a Vitrobot system (FEI
Company). Samples were imaged at ∼90 K in a JEOL 2100 TEM operating
at 200 kV. For normal TEM, solutions were deposited on hydrophilic
400 mesh carbon-coated copper grids. The micrographs shown are representative
for grid holes at different locations on the TEM grids and for samples
made on different days. TEM image analyses were done using custom
MATLAB scripts (The MathWorks Inc., Natick, MA, USA) and FIJI (https://fiji.sc/).

ten Hove J.B., Wang J., van Oosterom M.N., van Leeuwen F.W, & Velders A.H. (2017). Size-Sorting and Pattern Formation of Nanoparticle-Loaded Micellar Superstructures in Biconcave Thin Films. ACS Nano, 11(11), 11225-11231.

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Cryo-EM Imaging of GPI-TA Complex

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A total of 2.5 μL of the GPI-TA complex sample was applied to a glow-discharged holey carbon-film grid (Quantifoil, Au 1.2/1.3, 300 mesh) blotted with a Vitrobot system (FEI, Hillsboro, OR) using a 3.0 s blotting time with 100% humidity at 9 °C, then the sample was plunge-frozen in liquid ethane. Cryo-EM data collection was performed using a 300 kV Titan Krios microscope (FEI) equipped with a K2 summit direct electron detector camera (Gatan Inc., Pleasanton, CA, USA) set to super resolution mode, with a pixel size of 0.65 Å (a physical pixel size of 1.3 Å) and a defocus ranging from −1.5 µm to −2.3 µm. The specimen stage temperature was maintained at 80 K. The dose rate was 10 e⁻ s⁻¹, and each movie was 7.6 s long, dose-fractioned into 38 frames with an exposure of 1.18 e⁻ Å⁻² for each frame.

Liu S.S., Jin F., Liu Y.S., Murakami Y., Sugita Y., Kato T., Gao X.D., Kinoshita T., Hattori M, & Fujita M. (2021). Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit. Molecules, 26(18), 5462.

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Cryo-TEM Imaging of Lipoplexes and LNPs

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Cryo-TEM samples were prepared using 3 µL sample solutions of lipoplexes or LNPs (siRNA: 31 µM) at each N/P ratio. Total lipid concentrations of sample solutions were 17.4 mg/mL at an N/P ratio of 10 and 8.7 mg/mL at an N/P ratio of 5. Samples were added to a standard electron microscope grid with a perforated carbon film. Excess sample solution was removed from the grid by blotting and the grid was then plunge-frozen in liquid ethane to freeze the sample rapidly using a Vitrobot system (FEI, Hillsboro, OR, USA). Images were taken under cryogenic conditions (~88K) at a magnification of 50,000× with an AMT HR CCD camera (Advanced Microscopy Techniques corp., MA, USA). Samples were loaded with a Gatan 70° cryo-transfer holder in an FEI G20 Lab6 200 kV TEM (FEI) under low-dose conditions at an under focus of 4–6 µm to enhance image contrast. Experiments were performed at the Research Center for Ultra-High Voltage Electron Microscopy, Osaka University.

Kubota K., Onishi K., Sawaki K., Li T., Mitsuoka K., Sato T, & Takeoka S. (2017). Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation. International Journal of Nanomedicine, 12, 5121-5133.

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Cryo-EM Imaging of Liposome Structures

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The liposome structures were examined by the FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). A 200-mesh copper grid-supported holey carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA) was glow-discharged in an argon/oxygen atmosphere for 15 s. Four microliters of the liposome sample containing ~0.5 mM lipids were pipetted onto the copper grid, paper-blotted for 3 s in a 100% humidified chamber at 4 °C and plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot system (FEI, Hillsboro, OR, USA). The grids were stored in liquid nitrogen until mounted for imaging. EM imaging was conducted in bright-field mode at an operating voltage of 200 kV. Images were recorded at a defocus value of ~1.8 μm under low-dose exposures (25–30 e/Å²) with a 4k

\times

4k charge-coupled device camera (Glatan, Pleasanton, CA, USA) at a magnification of 50,000×. All experiments were carried out at the Academia Sinica Cryo-EM Facility (Taipei, Taiwan).

Jian C.B., Yu X.E., Gao H.D., Chen H.A., Jheng R.H., Chen C.Y, & Lee H.M. (2022). Liposomal PHD2 Inhibitors and the Enhanced Efficacy in Stabilizing HIF-1α. Nanomaterials, 12(1), 163.

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Cryogenic FilP Filament Imaging

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In standard assembly buffer, freshly dialyzed 200 μg/ml of FilP filaments were mixed with 1.8 nm Ni-NTA-Nanogold (Nanoprobes) or 10 nm gold fiducials for tomography. A drop of 4 μl of the sample was applied to 2/1 or 2/2 holy carbon film on 200 mesh copper grids (Quantifol) and vitrified by plunge freezing in liquid ethane using the Vitrobot system (FEI). Grids were transferred to the autoloader cassette to be analyzed with Titan Krios 300 kV cryo-TEM (FEI). Images were recorded with the K2 BioQuantum direct electron detector (Gatan). Nano-goldlabeled FilP images were collected at 130,000× magnification with a dose of 20 e⁻/Å². The model of nano-gold localization was created with Photoshop. Tilt series were collected at 35,000× magnification with 2° increment automatically with the Tomography software (FEI) over an angular range of −60° to +60°, with a total dose of ∼80 e⁻/Å². Frames were motion-corrected with MotionCorr2. 3D reconstruction, modeling, and visualization were completed with the IMOD software suite (Kremer et al, 1996 (link)).

Javadi A., Söderholm N., Olofsson A., Flärdh K, & Sandblad L. (2019). Assembly mechanisms of the bacterial cytoskeletal protein FilP. Life Science Alliance, 2(3), e201800290.

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Characterization of Nanoparticle Assemblies

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Dynamic Light Scattering (DLS) was done on a Malvern Zetasizer Nano S equipped with a laser operating at 633 nm. Sample grids for electron microscopy were obtained from Electron Microscopy Sciences (EMS, Hatfield, PA, USA) and were rendered hydrophilic using a plasma cleaning setup (~15 s at 10⁻¹ Torr). CryoTEM samples were cast on Quantifoil R2/2 grids. After blotting, samples were plunged into liquid ethane using a Vitrobot system (FEI Company). Samples were then imaged at ~90–100 K in a JEOL 2100 TEM operating at 200 kV or JEOL 1400Plus TEM operating at 120 kV. UV-Vis absorbance was evaluated to observe SPR shifts upon formation of micelle cores and concomitant approximation of the distance between gold nanoparticles; however, this was not evident (Fig. S13).

ten Hove J.B., van Oosterom M.N., van Leeuwen F.W, & Velders A.H. (2018). Nanoparticles reveal Extreme Size-Sorting and Morphologies in Complex Coacervate Superstructures. Scientific Reports, 8, 13820.

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Characterizing nanoparticles by DLS and cryoTEM

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Dynamic Light Scattering was done on a Malvern Zetasizer Nano S equipped with a laser operating at 633 nm. For cryoTEM, samples were cast on Quantifoil R2/2 grids or 400 mesh Holey Carbon grids. After blotting, samples were plunged into liquid ethane using a Vitrobot system (FEI Company). Samples were then imaged at ~90–100 K in a JEOL 2100 TEM operating at 200 kV or JEOL 1400Plus TEM operating at 120 kV. The electron dose was calibrated using the microscope’s software at the start of a cryoTEM session. Sample grids for electron microscopy were obtained from Electron Microscopy Sciences (EMS, Hatfield, PA, USA) and were rendered hydrophilic using a plasma cleaning setup (15 s at 10⁻¹ Torr). TEM image analyses were done using custom MATLAB scripts (The MathWorks Inc., USA) and FIJI (https://fiji.sc/).

ten Hove J.B., van Leeuwen F.W, & Velders A.H. (2018). Manipulating and monitoring nanoparticles in micellar thin film superstructures. Nature Communications, 9, 5207.

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Cryo-EM and Negative Stain of Budding Complexes

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For cryo-EM, 8 μl of 5 nm gold fiducials (BBI solutions) were added to 30 µl budding reaction mixture just prior to vitrification. Four microliters of budding reaction were applied to negatively glow-discharged lacey carbon grids (Agar Scientific, 200 Mesh Copper, S166) and plunge-frozen using an FEI Vitrobot system maintained at 4 °C with 100% humidity. Grids were stored in liquid nitrogen until use in EM.
For negative stain, 4 μl of budding reaction were applied to negatively glow-discharged continuous carbon film, 300 mesh, copper grids (EM Sciences, CF300-Cu), and stained with 2% uranyl acetate following standard procedures. Grids were viewed on the in-house T10 or T12 electron microscopes (Tecnai 100 and 120 kV, respectively) fitted with CCD cameras.

Hutchings J., Stancheva V., Miller E.A, & Zanetti G. (2018). Subtomogram averaging of COPII assemblies reveals how coat organization dictates membrane shape. Nature Communications, 9, 4154.

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Cryo-EM Imaging and Analysis of Liposomes

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Cryo-electron microscopy (EM) imaging and analysis of images was performed as described earlier (Chadda et al., 2018; (link)Cliff et al., 2019) (link). Briefly, liposomes were freeze-thawed seven times, and then extruded through a 400-nm nucleopore filter (GE Life Sciences) 21 times. 3 µL of the undiluted sample was loaded onto a glow-discharged Lacey carbon support film (Electron Microscope Sciences), blotted, and plunged into liquid ethane using a Vitrobot System (FEI). Images were collected on a FEI Titan Krios G3 300kV Cryo-TEM microscope with a Gatan K2 Summit Direct electron detector (GATAN). Magnifications of 6500x, 33 000x and 53 000x were used.
For size determination, liposomes were manually outlined in Fiji and ImageJ (Schindelin et al., 2012 (link)(Schindelin et al., , 2015) ) (link) to measure the outer radii of all liposomes, including those located on the carbon. Multilamellarity
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
was manually counted as the fraction of vesicles containing more than one bilayer. Liposome size distribution source data is provided in Fig. 4 -source data 1.

Chadda R., Bernhardt N., Kelley E.G., Teixeira S.C.M., Griffith K., Gil-Ley A., Öztürk T.N., Hughes L., Forsythe A., Krishnamani V., Faraldo‐Gómez J.D., & Robertson J. (2020). Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states.

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Cryo-TEM Analysis of Vesicle Bilayers

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Cryo-TEM was used for direct visualization of vesicle bilayers. To prepare samples for cryo-TEM analysis, Quantifoil copper R 1.2/1.3 200 mesh grids (Electron Microscopy Sciences) were prepared by glow discharging at 20 mA and 0.26 mbar for 1 min in a Pelco easiGlow system. Small volumes (2 μl) of the vesicle sample were applied to the carbon side of the EM grid in 90% humidity. Next, the liquid was blotted off for 0.5 s and the grids were plunge-frozen into precooled liquid ethane using a Vitrobot system (Thermo Scientific). This process enabled single vesicles to be embedded within a thin layer of amorphous ice, preserving them in their native state. The samples were then evaluated using a Thermo Scientific Glacios Cryo-TEM electron microscope. TEM images were acquired using an accelerating voltage of 200 kV and x120,000 magnification. The vesicle sizes were then measured using ImageJ.

Quinn S.D., Dresser L., Graham S., Conteduca D., Shepherd J, & Leake M.C. (2022). Crowding-induced morphological changes in synthetic lipid vesicles determined using smFRET. Frontiers in Bioengineering and Biotechnology, 10, 958026.

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