Tf20 microscope

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The TF20 microscope is a high-performance optical instrument designed for a wide range of imaging and analysis applications. It features advanced optics, precise control systems, and customizable configurations to meet the needs of various research and industrial settings.

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Lab products found in correlation

Em grid plunger, by Leica (2 mentions) Titan krios microscope, by Thermo Fisher Scientific (2 mentions) 2k 2k ccd camera, by Ametek (1 mentions) Vitrobot, by Thermo Fisher Scientific (1 mentions) Tecnai 12 microscope, by Ametek (1 mentions) Bm ultrascan, by Ametek (1 mentions) Falcon 2 camera, by Thermo Fisher Scientific (1 mentions) Em cpc manual plunger, by Leica (1 mentions) Sp2 confocal microscope, by Leica (1 mentions) Axis ultra dld x ray photoelectron spectrometer, by Shimadzu (1 mentions) Uv 2550 spectrophotometer, by Shimadzu (1 mentions) Fei vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)

8 protocols using tf20 microscope

Archaella Visualization Using Electron Microscopy

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Purified archaella (4 μl) were pipetted onto carbon-coated copper grids and allowed to settle for 1 min before blotting to remove excess sample. A 2 μl volume of 1% uranyl acetate was added for 1 min and excess stain was blotted away. Data were collected using the FEI TF20 microscope with a 16-megapixel charge-coupled device (CCD) camera and the automated imaging software Leginon^{53 (link)} (Supplementary Fig. 8a–c). For cryoEM samples, the concentration of the M. hungatei archaella was first optimized by negatively staining the sample with 1% uranyl acetate and imaged in an FEI TF12 microscope with a Gatan 2k×2k CCD camera. Samples were diluted as needed to achieve the appropriate sample concentration.

Poweleit N., Ge P., Nguyen H.H., Ogorzalek Loo R.R., Gunsalus R.P, & Zhou Z.H. (2016). CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pili. Nature microbiology, 2, 16222.

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Cryo-EM Sample Preparation and Imaging

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2 µl of sample was applied to 1.2/1.3 µm Quantifoild Grids (Electron Microscopy Sciences, Hatfield, PA), blotted, and frozen in liquid ethane with a Vitrobot (FEI). Images were recorded with a 2–4.5 µm defocus range using low dose procedures on a TF20 microscope (FEI) operating at 200 kV and recorded on a 8192 × 8192 CMOS camera (TVIPS, Gauting, Germany) at 62k magnification corresponding to a pixel size of 1.29 Å.

Booth D.S., Cheng Y, & Frankel A.D. (2014). The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA. eLife, 3, e04121.

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Cryo-EM Imaging of Dynamin Polymers

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Aliquots of 3.5 ul of each sample was applied to plasma-cleaned (Fishione Inc.) C-flat grids (Protochips, CF-1.2/1.3–4C), blotted on the sample side with filter paper for 2 seconds (22 °C, 90% humidity) and then plunged into liquid ethane with a Leica EM Grid Plunger (Leica Microsystems). For the dyn^GTP samples, after 3.5 ul sample was applied to the grids in the grid plunger, GTP was added and plunged into ethane after 5–10 seconds. The vitrified samples were stored in liquid nitrogen before examination by cryo-EM. For the dyn^GMPPCP polymer samples, images were recorded during three sessions on a Titan Krios microscope (FEI) at 300 kV and recorded at 22,500X magnification with a defocus range of 1.0–3.0 μm on a K2 summit camera in counting mode. For the GMPPCP treated sample containing partially constricted polymers and for the dyn^GTP sample, images were recorded on a TF20 microscope (FEI) at 200 kV and recorded at 29,000X magnification, with a defocus range of 1.5–3.0 μm on a K2 summit camera in counting mode (Extended Data Table 1).

Kong L., Sochacki K.A., Wang H., Fang S., Canagarajah B., Kehr A.D., Rice W.J., Strub M.P., Taraska J.W, & Hinshaw J.E. (2018). Cryo-EM of the dynamin polymer assembled on lipid membrane. Nature, 560(7717), 258-262.

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Cryo-EM Sample Preparation and Optimization

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For EM grid preparation, the CCNC cryo-EM sample was then dialyzed in 10 mM HEPES, (pH 7.5), 50 mM NaCl, 1 mM EDTA and 1 mM DTT and was diluted with an appropriate volume of the dialysis buffer supplemented with or without NP-40 (0.004%). Negative stain analysis was performed to identify conditions that yielded potentially appropriate concentration, homogeneity, and homogenous distribution of particles for subsequent cryo-EM grid preparations. To do this, 3 µl (0.03 mg/ml) of dialyzed complex was loaded on glow-discharged 300 mesh Cu carbon grids (EMS) and incubated for 30 s before staining with 2% uranyl acetate. All samples were imaged on a FEI Tecnai 12 microscope operating at 120 kV, using a CCD camera (Gatan BM-Ultrascan) at a nominal magnification of 39,000x (Figure S2B). To prepare cryo-EM grids, 2 µl of the dialyzed CCNC (~0.06 and ~0.5 mg/mL without and with 0.004% NP-40) was applied to CF1.2/1.3 holey carbon grids that were glow discharged for 40 s before sample application, subsequently blotted for 8 s, and flash-frozen by plunging into liquid ethane with a Leica EM CPC manual plunger. EM grids were made in batches and freezing conditions were optimized using an FEI TF20 microscope operating at 200 kV equipped with a FEI Falcon II camera.

Allu P.K., Dawicki-McKenna J.M., Eeuwen T.V., Slavin M., Braitbard M., Xu C., Kalisman N., Murakami K, & Black B.E. (2019). Structure of the Human Core Centromeric Nucleosome Complex. Current biology : CB, 29(16), 2625-2639.e5.

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Cryo-EM Analysis of RBD-Foldon

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Purified RBD-foldon in 4 μl was applied to a glow-discharged copper grid overlaid with formvar and amorphous carbon (Ted Pella). Negative staining was performed with Nano-W organotungstate stain (Nanoprobes) according to the manufacturer's protocol. The sample imaged using an FEI TF-20 microscope operating at 200 kV, with a magnification of 62,000× and defocus of -2.5 μm. Micrographs were contrast transfer function (CTF)-corrected in RELION using CTFFIND-4.1 44 . A Article small manually picked dataset was used to generate 2D references for autopicking. The resulting particle set was subjected to 2D classification in RELION 3.0.6 45 .

Vogel A.B., Kanevsky I., Che Y., Swanson K.A., Muik A., Vormehr M., Kranz L.M., Walzer K.C., Hein S., Güler A., Loschko J., Maddur M.S., Ota-Setlik A., Tompkins K., Cole J., Lui B.G., Ziegenhals T., Plaschke A., Eisel D., Dany S.C., Fesser S., Erbar S., Bates F., Schneider D., Jesionek B., Sänger B., Wallisch A.K., Feuchter Y., Junginger H., Krumm S.A., Heinen A.P., Adams-Quack P., Schlereth J., Schille S., Kröner C., de la Caridad Güimil Garcia R., Hiller T., Fischer L., Sellers R.S., Choudhary S., Gonzalez O., Vascotto F., Gutman M.R., Fontenot J.A., Hall-Ursone S., Brasky K., Griffor M.C., Han S., Su A.A.H., Lees J.A., Nedoma N.L., Mashalidis E.H., Sahasrabudhe P.V., Tan C.Y., Pavliakova D., Singh G., Fontes-Garfias C., Pride M., Scully I.L., Ciolino T., Obregon J., Gazi M., Carrion R J.r., Alfson K.J., Kalina W.V., Kaushal D., Shi P.Y., Klamp T., Rosenbaum C., Kuhn A.N., Türeci Ö., Dormitzer P.R., Jansen K.U, & Sahin U. (2021). BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature, 592(7853).

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Cryo-EM Imaging of Dynamin Polymers

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Multimodal Analysis of Nanoparticle Properties

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High-resolution transmission electron microscopy (HRTEM) images were taken on a FEI TF-20 microscope (FEI, Hillsboro, PerkinElmer, Boston, USA). The fluorescence spectra were carried out on an LS-55 fluorescence spectrometer (Nicolet Co., Madison, WI, USA). The Fourier transform infrared (FT-IR) spectrum was obtained with a Magna-IR560 FT-IR spectrometer (Nicolet Co., Madison, WI, USA) within the range of 400–4000 cm⁻¹, while the UV-vis absorption spectra were performed on a UV-2550 spectrophotometer (Shimadzu, Kratos, Japan). The X-ray photoelectron spectroscopy (XPS) images were captured on an AXIS ULTRA DLD X-ray photoelectron spectrometer (Kratos, Manchester, UK). The fluorescence cell images were performed with a Leica SP2 confocal microscope.

Shi Y., Sun C., Gao X., Zhao W, & Zhou N. (2019). Sensitively and Selectively Detect Biothiols by Using Fluorescence Method and Resonance Light Scattering Technique Simultaneously. Molecules, 24(22), 4136.

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Cryo-EM Visualization of Extracellular Vesicles

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Extracellular vesicles were flash frozen and stored at -80 C until use. Prior to preparing cryo-EM grids, the sample was diluted to 1.5 mg/ml as determined by a Bradford assay. C-flat grids (Copper, 1.2/1.3, Protochips) were prepared by glow-discharged for 30 sec in a PELCO Easiglow glow-discharge unit at 15 mA. We applied 3 l of EV in PBS to the grid and incubated for 60 sec before vitrification using an FEI Vitrobot Mark IV (ThermoFisher). The grids were blotted for 3 sec using blotting force 3 at 4 C and ~90% humidity and plunged in liquid ethane.
Images were collected using a 626 Gatan cryo-holder on a TF20 microscope (FEI) equipped with a K2 Summit (Gatan) direct detector. We automated the collection of data using SerialEM (Mastronarde, 2005) . 60 movies (50 frames/movie) of each sample were acquired over 6.8 sec with an exposure rate of 14.0 e -/pix/sec, yielding a total dose of 56 e - /Å 2 , and a nominal defocus range from -1.0 μm to -3.0 μm. Images were gain corrected using the method described by (Afanasyev et al., 2015) and motion-corrected in cisTEM (Grant et al., 2018) . We measured the length and width of EVs in 20 consecutively collected images per sample manually in tdisp (https://sourceforge.net/projects/tigris/) and calculated means and standard deviations of counted vesicles per image.

Barragán‐Iglesias P., Peña J.B.d.l., Lou T., Loerch S., Kunder N., Shukla T., Basavarajappa L., Song J., Megat S., Moy J.K., Wanghzou A., Ray P.R., Shepherd J.D., Hoyt K., Steward O., Price T.J., & Campbell Z.T. (2020). Intercellular Arc signaling regulates vasodilation.

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