Autogrid

Manufactured by Thermo Fisher Scientific

Sourced in Japan, Netherlands, United Kingdom

Autogrids is a laboratory equipment product designed for the automated preparation of samples. It provides a standardized and efficient process for sample preparation, ensuring consistent results across multiple samples.

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Lab products found in correlation

22 protocols using autogrid

Single-Particle Cryo-EM Imaging Protocol

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Using the auto-grid adapter cartridge (JEOL, Tokyo, Japan), a single pre-screened cryo-grid clipped as an auto-grid (Thermo Fisher Scientific) was transferred to a CRYO ARM 300 (JEOL) transmission cryo-electron microscope operated at 300 kV and equipped with a cold-field emission gun and an in-column Ω filter. Image acquisition was performed using flood beam parallel illumination in bright field imaging mode. Movies were automatically recorded by SerialEM using image shift (5 × 5 holes per stage position) and a K3 Direct Detection Camera (Gatan, AMETEK, Pleasanton, CA, USA) in CDS mode at a nominal magnification of ×60,000 at the camera level, corresponding to a pixel size of 0.806 Å with 48 frames in 3 s exposure time, resulting in a total dose of 48 e⁻/Å². A total of 3018 movies were collected in series within a defocus range of −0.5 μm to −1.5 μm. A typical micrograph and angular distribution of protein complexes are shown in Supplementary Figs. 2b and 3b, respectively.

Li J., Hamaoka N., Makino F., Kawamoto A., Lin Y., Rögner M., Nowaczyk M.M., Lee Y.H., Namba K., Gerle C, & Kurisu G. (2022). Structure of cyanobacterial photosystem I complexed with ferredoxin at 1.97 Å resolution. Communications Biology, 5, 951.

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Cryo-EM Grid Preparation for FIB-SEM

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The crystal slurry was diluted to a final concentration of 10% v/v and applied to UltrAuFoil Gold 200 mesh 2/2 cryo-EM grids (Storm et al., 2020 ▸ ). Prior to loading in the FIB-SEM, the grids were mounted in specific FIB-compatible AutoGrid rings and secured with a c-clip (Thermo Fisher Scientific).

Parkhurst J.M., Crawshaw A.D., Siebert C.A., Dumoux M., Owen C.D., Nunes P., Waterman D., Glen T., Stuart D.I., Naismith J.H, & Evans G. (2023). Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae. IUCrJ, 10(Pt 3), 270-287.

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Shuttle Holder Mounting and Temperature Control

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The sample shuttle holder mounts to the cold finger protruding at the front of the microcooler. Two temperature sensors (Pt-1000) are present, one residing on the shuttle holder (

T_{s h}

) and one placed on the micro cold stage (

T_{c s}

). Near each sensor, a surface-mount technology-based resistor (68 Ω, International Manufacturing Services #IMS004-3-68) is placed acting as a heater. The thermal gradient between shuttle holder and micro cold stage is ∼5 K, allowing the temperature of both to be regulated semi-independently through control loops. The shuttle contains the AutoGrid (Thermo Fisher Scientific) in which the TEM grid is held in place by a c-clip. Starting with an empty shuttle, first the coverslip (ITO-coated D263M glass, 3.4 mm diameter, Schott AG) is placed, followed by the AutoGrid, spacer ring and screw. The shuttle has an actuated leaf spring mechanism which is counteracted when the shuttle is picked up by the transfer rod via a threaded interface. With the transfer rod fully engaged, the leaf spring folds around the main body to reduce the shuttle width so it can move inside the shuttle holder. When unscrewing the transfer rod, the compression spring pushes the leaf spring mechanism outwards, effectively clamping the shuttle inside the holder to ensure good thermal contact between holder and shuttle.

Boltje D.B., Hoogenboom J.P., Jakobi A.J., Jensen G.J., Jonker C.T., Kaag M.J., Koster A.J., Last M.G., de Agrela Pinto C., Plitzko J.M., Raunser S., Tacke S., Wang Z., van der Wee E.B., Wepf R, & den Hoedt S. (2022). A cryogenic, coincident fluorescence, electron, and ion beam microscope. eLife, 11, e82891.

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Cryo-Electron Tomography Workflow

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The 200-nm-thick sections were cut on a Leica UC6 ultramicrotome. Grids were assembled into an Autogrid (Thermo Fisher) and loaded onto a 200 kV Thermo Fisher Talos Arctica fitted with a Falcon 3EC (Thermo Fisher) camera operated in linear mode and at room temperature (RT). Bidirectional dual axis tilt series were acquired at 1° increments from −60° to +60° under the control of Tomography software (Thermo Fisher). Tilt series were reconstructed using weighted back-projection with IMOD.

Rae J., Ferguson C., Ariotti N., Webb R.I., Cheng H.H., Mead J.L., Riches J.D., Hunter D.J., Martel N., Baltos J., Christopoulos A., Bryce N.S., Cagigas M.L., Fonseka S., Sayre M.E., Hardeman E.C., Gunning P.W., Gambin Y., Hall T.E, & Parton R.G. (2021). A robust method for particulate detection of a genetic tag for 3D electron microscopy. eLife, 10, e64630.

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Cryo-FIB Milling of Muscle Fiber

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The vitrified grid containing the isolated muscle fiber was clipped into a special AutoGrid (Thermo Fisher Scientific) designed for cryo-FIB milling. After being loaded into the transfer shuttle (79 (link)), the grid was transferred into a cryo-FIB/scanning electron microscopy (SEM) dual-beam microscope Aquilos2 (Thermo Fisher Scientific). Before milling, the grid was sputter-coated with platinum for 10 s to reduce the charging effects of the sample. Then, it was injected with organometallic platinum for 12 s to prevent damage by gallium ion beam milling. The grid was pretilted 8° to expose the muscle fibers on the grid. The sample was milled to ~300 nm following the regular steps. Then, the cryo-lamella was polished further using a 10-pA current and examined using cryo-SEM images to estimate the thickness by charging propensity. The process was continued until the thickness reached ~120 nm.

Xu J., Liao C., Yin C.C., Li G., Zhu Y, & Sun F. (2024). In situ structural insights into the excitation-contraction coupling mechanism of skeletal muscle. Science Advances, 10(12), eadl1126.

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Proteinase K Crystal Milling and Lamellae Preparation

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Milling of proteinase K crystals was carried out as previously described (Schaffer et al., 2015 , 2017 (link); Duyvesteyn et al., 2018 (link)) using a Scios^TM DualBeam^TM cryoFIB microscope (Thermo Fisher Scientific) equipped with a Quorum PP3010T cryotransfer system and a Quorum cryostage and shuttle. Briefly, plunge-frozen grids containing crystals of proteinase K with approximate dimensions 12 × 10 × 10 μm were loaded into autogrid (Thermo Fisher Scientific) compatible with FIB-SEM applications. The grids were then coated with an organoplatinum compound using the in situ gas injection system (GIS) of the cryoFIB instrument. Lamellae were generated through a series of milling steps, where the current of the Ga beam was decreased in a stepwise fashion from 300 to 30 pA. These steps corresponded to subsequent lamella thicknesses of approximately 5 μm down to 0.2 μm, respectively. In addition to the initial organoplatinum coating, the sample was sputter coated with metallic platinum post-milling using the Quorum PP3010T system (10 mA, 3 s, argon atmosphere) to reduce beam-induced charging effects.

Beale E.V., Waterman D.G., Hecksel C., van Rooyen J., Gilchrist J.B., Parkhurst J.M., de Haas F., Buijsse B., Evans G, & Zhang P. (2020). A Workflow for Protein Structure Determination From Thin Crystal Lamella by Micro-Electron Diffraction. Frontiers in Molecular Biosciences, 7, 179.

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Cryo-EM Tomography Acquisition Protocol

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Thick 200 nm sections were cut on a Leica UC6 ultramicrotome. Grids were assembled into an Autogrid (Thermo Fisher) and loaded onto a 200 kV Thermo Fisher Talos Arctica fitted with a Falcon 3EC (Thermo Fisher) camera operated in linear mode and at room temperature.
Bidirectional dual axis tilt series were acquired at 1 o increments from -60 o to +60 o under the control of Tomography software (Thermo Fisher). Tilt series were reconstructed using weighted-back projection with IMOD.

Rae J., Ferguson C., Ariotti N., Webb R.I., Cheng H., Mead J.L., Riches J.D., Hunter D.J.B., Martel N., Baltos J., Christopoulos A., Bryce N.S., Cagigas M.L., Fonseka S., Hardeman E.C., Gunning P.W., Gambin Y., Hall T.E., & Parton R.G. (2020). APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy.

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Cryo-EM Analysis of Cell Envelopes

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Quantifoil R2/2 holey carbon grids were glow-discharged prior use. Both samples, the isolated cell envelopes and the isolated protein, were blotted and vitrified using a Vitrobot plunge-freezing machine (Mark IV, ThermoFisher) at room temperature (blot force 0, blotting time 3 s, 100% humidity), and placed in autogrid (FEI, Eindhoven, Netherlands) prior image acquisition.
Micrographs were acquired with a Titan-Krios TEM (ThermoFisher) at an operating voltage of 300 kV and equipped with a Cs-corrector (cs 2.7 mm), a Quantum GIF energy filter (slit width set to 20 eV), and a post-GIF K2 camera (Gatan) in counting mode. Images were manually recorded by the EPU software at a nominal magnification of 33.000×, yielding a final image pixel size of 4.37 Å. Image defocus was set at − 3.0 μm, the total electron dose used to acquire a single image was ~ 40 electrons/Å².

Farci D., Cocco E., Tanas M., Kirkpatrick J., Maxia A., Tamburini E., Schröder W.P, & Piano D. (2022). Isolation and characterization of a main porin from the outer membrane of Salinibacter ruber. Journal of Bioenergetics and Biomembranes, 54(5-6), 273-281.

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Cryo-EM Tomography of Cell Envelope

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The grid preparation and data acquisition were done at Central European Institute of Technology, Brno, Czech Republic. Quantifoil R2/1.3 holey carbon grids (Quantifoil Micro Tools GmbH, Germany) were glow discharged prior to use. Cell-envelope samples were fast frozen into liquid ethane using a Vitrobot Mark IV (ThermoFisher, the Netherlands) plunge-freezing machine (4 °C, 100% humidity, blot force 2, blotting time 2 s) and placed in autogrid (FEI, Eindhoven, the Netherlands) prior to image acquisition. Grids were transferred to a Titan Krios transmission EM (TEM) (ThermoFisher, the Netherlands) operating at 300 kV and equipped with a Cs corrector (cs 2.7 mm), a Quantum GIF energy filter (slit width set to 20 eV), and a post-GIF K2 camera (Gatan, USA) at a magnification corresponding to a pixel size of 2.28 Å/px (∼21,000×). A dose-symmetric tilt scheme with a 3° increment (0, +3°, −3°, etc.; tilting range ± 60°) and a total dose of 2 e⁻/Å² were used for tomography acquisition by SerialEM software (40 (link)). The defocus was set to vary between 2 µm and 5 µm.

Farci D., Haniewicz P, & Piano D. (2022). The structured organization of Deinococcus radiodurans’ cell envelope. Proceedings of the National Academy of Sciences of the United States of America, 119(45), e2209111119.

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Cryo-FIB/SEM Lamellae Preparation

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Grids were mounted into modified Autogrids (ThermoFisher) for mechanical support. Clipped grids were loaded into an Aquilos (ThermoFisher) dual‐beam cryo‐focused ion beam/scanning electron microscope (cryo‐FIB/SEM). All SEM imaging was performed at 2 kV and 13 pA, whereas FIB imaging for targeting was performed at 30 kV and 10 pA. Milling was typically performed with a stage tilt of 18°, so lamellae were inclined 11° relative to the grid. Each lamella was milled in four stages: an initial rough mill at 1 nA beam current, an intermediate mill at 300 pA, a fine mill at 100 pA, and a polishing step at 30 pA. Lamellae were milled with the wedge pre‐milling technique (Schaffer et al,2017) and with expansion segments (Wolff et al,2019).

Leung M.R., Roelofs M.C., Ravi R.T., Maitan P., Henning H., Zhang M., Bromfield E.G., Howes S.C., Gadella B.M., Bloomfield‐Gadêlha H, & Zeev‐Ben‐Mordehai T. (2021). The multi‐scale architecture of mammalian sperm flagella and implications for ciliary motility. The EMBO Journal, 40(7), e107410.

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