Falcon 2 direct electron detector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Falcon II direct electron detector is a high-performance imaging device designed for use in electron microscopy. It is capable of capturing high-resolution, low-noise images by directly detecting the electron beam. The Falcon II provides efficient conversion of electron signals into digital data, enabling researchers to obtain detailed information about the structure and composition of their samples.

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Lab products found in correlation

32 protocols using falcon 2 direct electron detector

Cryo-EM Sample Preparation and Data Collection

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Samples were prepared by pipetting a 3 μl sample, mixed with a gold fiducial solution (15 nm gold particle size), onto a freshly glow-discharged Lacey carbon grids (Ted Pella Inc., Redding, CA, USA). The grids were plunge-frozen in liquid ethane in a Vitrobot Mark IV (FEI, Eindhoven, The Netherlands). Cryo-EM data were collected using a Titan Krios microscope equipped with Falcon II direct electron detector (FEI, Eindhoven, The Netherlands), operated at 300 kV. Low-dose imaging conditions with 20 electrons per Å² were applied. Images were collected at 1–3 μm underfocus. All micrographs were recorded at a pixel size of 1.34 Å per pixel.
Tilt series data were obtained on a Titan Krios (FEI, Eindhoven, The Netherlands), operated at 300 kV on a Falcon II direct electron detector (FEI, Eindhoven, The Netherlands), under low-dose conditions with 50 electrons per Å². Tomograms of selected areas were obtained from -70 to +70 degrees with a 1.5° tilt increment. The defocus range of the data set was 5–8 μm, with a pixel size of 6 Å. The tilted images were aligned and reconstructed with both the FEI Inspect3D software (FEI, Eindhoven, The Netherlands) and with Tomo3D [42 (link)] and the SIRT reconstruction method (30 iterations).

Vázquez-Fernández E., Vos M.R., Afanasyev P., Cebey L., Sevillano A.M., Vidal E., Rosa I., Renault L., Ramos A., Peters P.J., Fernández J.J., van Heel M., Young H.S., Requena J.R, & Wille H. (2016). The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy. PLoS Pathogens, 12(9), e1005835.

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High-resolution Cryo-EM Imaging

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Grids were imaged on an F20 microscope (FEI) equipped with a Falcon II direct electron detector (FEI) at 200 kV. Images were collected at a dose of 30–35 e⁻ Å⁻², a magnification of 62,000-fold (1.8 Å pixel size), and a defocus range from −1.8 to −3.3 µm.

Edwardson T.G., Tetter S, & Hilvert D. (2020). Two-tier supramolecular encapsulation of small molecules in a protein cage. Nature Communications, 11, 5410.

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Negative and Positive Stain TEM Imaging

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Data collection of negative stain grids was performed on a Tecnai LaB6 G² Spirit transmission electron microscope (FEI) operating at 120 keV (EM STP, The Francis Crick institute). Micrographs were collected using a 2Kx2K GATAN Ultrascan 100 camera at a nominal magnification of 30,000 (3.45 Å pixel size) or 21,000 (4.92 Å pixel size, train classes) within a -0.5 to 2.5 μm defocus range. Analysis of positive stain grids was performed on a Tecnai G² F20 TWIN electron microscope operating at 200 keV (FEI; Electron Microscopy Centre, Imperial College London) equipped with a Falcon II direct electron detector (FEI). Micrographs were collected at a nominal magnification of 50,000 (2.05 Å pixel size) in a defocus range from -3 to -6 μm.

Douglas M.E., Ali F.A., Costa A, & Diffley J.F. (2018). The mechanism of eukaryotic CMG helicase activation. Nature, 555(7695), 265-268.

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Single-particle cryo-EM of IMV

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R0.6/1.0 Quantifoil grids (Quantifoil Micro Tools, Jena, Germany) were glow discharged and pretreated with a solution of 10-nm colloidal gold particles (MP Biomedicals, USA). A 2.6-µl solution of purified IMV was applied to the grid. The grids were blotted briefly with filter paper and then were rapidly plunged into liquid ethane using Vitrobot Mark II (FEI) for freezing. Electron microscopy images were collected at the liquid nitrogen temperature using a Titan Krios electron microscope (FEI) equipped with a field emission gun and a Falcon II direct electron detector (FEI). The microscope was operated at 300 kV and a nominal magnification of ×37,000 with a calibrated pixel size of 4.46 Å after 2 × 2 binning. Images of single-axis tilt series were collected covering an angular range from −70° to +70° with a nonlinear Saxton tilt scheme at 4- to 7-µm underfocus using the Xplore three-dimensional (3D) software package (FEI) and a cumulative dose of ~120 e⁻/Å². The IMOD package (50 (link)) was used to align tilted projection images and to generate the final 3D density map from the aligned image stack. The final 3D density map was obtained by the simultaneous iterative reconstruction technique (SIRT).

Terashima H., Kawamoto A., Tatsumi C., Namba K., Minamino T, & Imada K. (2018). In Vitro Reconstitution of Functional Type III Protein Export and Insights into Flagellar Assembly. mBio, 9(3), e00988-18.

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Structural Characterization of PSII-LHCII Supercomplex

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PSII-LHCII supercomplexes were prepared at 1 mg mL⁻¹ in the sucrose gradient buffer. 4 μL of sample were applied to a glow-discharged lacey carbon grid (200 Cu mesh, Quantifoil) within the chamber of a Vitrobot (mark 3, FEI). After 60 s incubation at 100% humidity and 21 °C, a quick wash with 4 μL of 10 mM Hepes pH 7.5 was performed to remove sugar, excess solution was blotted from both sides for 4 s, and the grid was plunge-frozen in a liquid ethane/propane mixture. Samples were exposed to only dim green light during the grid preparation procedure. Data collection was performed on a Titan Krios microscope (FEI) operated at 300 kV using EPU automated acquisition software (FEI). Spanning a defocus range −1 to −3 μm, 6,834 micrographs were recorded on a Falcon II direct electron detector (FEI) at 59,000 magnification (image pixel size of 1.4 Å), with a total dose of 47.5 e⁻/Å² fractionated over 7 frames (1.5 s exposure, dose rate of 30 e⁻/Å²/s).

Albanese P., Melero R., Engel B.D., Grinzato A., Berto P., Manfredi M., Chiodoni A., Vargas J., Sorzano C.Ó., Marengo E., Saracco G., Zanotti G., Carazo J.M, & Pagliano C. (2017). Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap. Scientific Reports, 7, 10067.

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Visualizing HAP2e Protein in Liposomes

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Purified HAP2e (C. reinhardtii) mixed with liposomes was spotted on glow discharged carbon grids (CF300, EMS, USA), negatively stained with 2% phosphotungstic acid (PTA) pH 7.4, analyzed with a Tecnai G2 Bio-Twin electron microscope (FEI, USA) and imaged with an Eagle camera (FEI, USA). For cryo-electron microscopy liposomes alone or liposomes mixed with purified HAP2e were applied on a glow discharged Lacey Carbon grid (Agar Scientific, UK). Samples were plunge-frozen in liquid ethane using an automated system (Leica EMGP, Austria) and visualized on a Tecnai F20 electron microscope operating at a voltage of 200 kV. Image frames were recorded in low-dose mode on a Falcon II direct electron detector (FEI, USA).

Fédry J., Liu Y., Péhau-Arnaudet G., Pei J., Li W., Tortorici M.A., Traincard F., Meola A., Bricogne G., Grishin N.V., Snell W.J., Rey F.A, & Krey T. (2017). The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein. Cell, 168(5), 904-915.e10.

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Cryo-EM Imaging of Bacterial Membranes

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Strains were grown aerobically in LB at 37°C until an OD₆₀₀ of 0.6 was reached. Cells were spun for 5 min at 6,000 × g at 4°C and resuspended to an OD₆₀₀ of about 12.
UltraAuFoil R2/2 grids (200 mesh; Quantifoil Micro Tools GmbH) were glow-discharged for 60 s at 10 mA. Cells were mixed with a solution of 10 nm colloidal gold (Sigma) immediately before freezing. A 2.5-μl droplet of sample was applied to the grid and plunge frozen using a Vitrobot MkIV (FEI Company) with a wait time of 60 s, a blot time of 5 s, a blot force of 3, and a drain time of 1 s at a constant humidity of 100%. Grids were stored under liquid nitrogen until required for data collection.
Projection images were collected on a 200 keV FEI Tecnai TF20 FEG transmission electron microscope (FEI Company) equipped with a Falcon II direct electron detector (FEI Company) using a Gatan 626 cryogenic-holder (Gatan). Leginon automated data-collection software 3.0 [41 (link)] was used to acquire images with pixel size of 0.828 nm (nominal magnification 25,000×) with a defocus of −5 μm. Membrane measurements were carried out as previously described [25 (link)]. Briefly, 3dmod from the IMOD package [42 (link)] and custom scripts were used to manually segment the IMs and OMs of projection images of about 35 cells per mutant, measuring the periplasmic width at 0.5-nm intervals to produce width histograms (Fig 2B).

Asmar A.T., Ferreira J.L., Cohen E.J., Cho S.H., Beeby M., Hughes K.T, & Collet J.F. (2017). Communication across the bacterial cell envelope depends on the size of the periplasm. PLoS Biology, 15(12), e2004303.

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Visualizing CmDnm1 Protein Assembly

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Prior to visualization, proteins were diluted to indicated concentrations in a buffer containing 25 mM HEPES, 150 mM sodium chloride, and 2 mM magnesium sulphate, final pH 7.5. For tubulation, 10 μM CmDnm1 was incubated at 22 °C for 2 hrs with 2 mg/ml liposomes. Ring formation was achieved at 8 μM concentration whilst single particles were observed at 0.3 – 1 μM concentration. 1 mM nucleotide was added as desired. The samples were applied to glow-discharged 300-mesh carbon coated copper grids and stained with 2 % uranyl acetate. Images were recorded on either a FEI Tecnai T12 electron microscope with CCD camera, or a FEI Tecnai TF20 FEG electron microscope equipped with a Falcon II direct electron detector (FEI Company). To generate class averages of CmDnm1 rings, datasets were collected between -2 and -3.5 defocus with 2.05 Å pixel size. To generate class averages of CmDnm1 single particles, a dataset was collected between -1.5 and -3.5 defocus with 1.28 Å pixel size. Images were phase flipped using GCTF and initially processed by iterative rounds of 2D classification using RELION to remove low quality particles. This resulted in stacks containing 6806 (apo) and 14,573 (GMPPCP) images for the rings, and 7740 images for the single particles. Multivariate statistical analysis and 2D classification in IMAGIC was then carried out to generate the final class averages.

Bohuszewicz O, & Low H.H. (2018). Structure of a mitochondrial fission dynamin in the closed conformation. Nature structural & molecular biology, 25(8), 722-731.

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Structural Determination of Hoxa9 IRES-80S Complex

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5 μl of the Hoxa9 IRES-80S complex were applied for 30 sec to glow discharged R2/2 holey carbon grids (Quantifoil Micro Tools, Großlöbichau) which was coated with a thin film of carbon using a BAE 120 thin-film coating system (Balzers, Pfäffikon). Excess liquid was blotted away for 12 sec and the grid were frozen in liquid ethane/propane (1:2) using a Vitrobot Mk IV (FEI Company, Hillsboro) at 4°C and 100% relative humidity. The grids were imaged in a Titan Krios cryo-electron microscope (FEI Company, Hillsboro) at 300 kV and a magnification of 100 720 × with a Falcon II direct electron detector (FEI Company, Hillsboro). Micrographs were recorded using dose fractionation with 37 frames and a total dose of 40 e⁻ /A² with the EPU software for automated data collection. Defocus values of the micrographs in the final dataset range from −700 nm to −3800 nm.

Leppek K., Fujii K., Quade N., Susanto T.T., Boehringer D., Lenarčič T., Xue S., Genuth N.R., Ban N, & Barna M. (2020). Gene- and species-specific Hox mRNA translation by ribosome expansion segments. Molecular cell, 80(6), 980-995.e13.

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Cryo-EM Grid Preparation and Imaging

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3 μl of each complex were applied onto glow-discharged Quantifoil R2/2 cryo-EM grids covered with continuous carbon (of around 50 Å thick) at 4°C and 100% ambient humidity. After a 30 s incubation, the grids were blotted for 3-3.5 s and vitrified in liquid ethane using a Vitrobot Mk3 (FEI).
Automated data acquisitions (EPU software, FEI) were done on Tecnai F30 Polara and Titan Krios microscopes (FEI) at 300 kV for the Sample1 dataset and the Sample2 (IF2-containing dataset), respectively. For the Sample1 dataset, images of 1.1 s/exposure and 17 movie frames were recorded on a Falcon III direct electron detector (FEI) at a calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). For the Sample2 dataset, images of 1.5 s/exposure and 25 movie frames were recorded on a Falcon II direct electron detector (FEI) at a calibrated magnification of 104,478, resulting in a pixel size of 1.34 Å. For both datasets, dose rates of 27-30 electrons per Å² per second and ranges from 1.5 to 3.0 μm defocus values were used. Micrographs that showed noticeable signs of astigmatism or drift were discarded.

Hussain T., Llácer J.L., Wimberly B.T., Kieft J.S, & Ramakrishnan V. (2016). Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation. Cell, 167(1), 133-144.e13.

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