Infected BBEC cultures representing selected time points p.i. were fixed and processed for TEM as previously described (59 (link)). The cultures were analyzed on a FEI Tecnai transmission electron microscope at 200 kV and images captured with a Gatan Multiscan 794 camera.
Multiscan 794
Manufactured by Ametek
Sourced in United States
The MultiScan 794 is a laboratory equipment product designed for multi-channel data acquisition and analysis. It features high-speed data sampling and a modular architecture to support a variety of sensor inputs. The core function of the MultiScan 794 is to capture and process sensor data across multiple channels simultaneously.
Automatically generated - may contain errors
4 protocols using multiscan 794
1
Ultrastructural Analysis of Infected Cells
2
Transmission Electron Microscopy of C. merolae
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Samples of an induced C. merolae culture were gently pelleted and resuspended in cacodylate buffer (75 mM cacodylate, pH 7.0) supplemented with 2% glutaraldehyde. Samples were fixed for 3.5 h on ice. Cells were immobilised in 2% agarose in cacodylate buffer before fixing with 1% OsO4 overnight at 4 °C. After washing with cacodylate buffer three more times, samples were dehydrated through a graded series of acetone in cacodylate buffer (30, 50, 70, 90, 100%) at 4 °C with two additional changes in the 100% at room temperature. Cells were embedded into epoxy resin (ERL-4221D, D.E.R 736, nonenyl succinic anhydride, dimethylaminoethanol) following a protocol by Spurr [36 (link)].
Ultra-thin sections (~80 nm) were prepared on a microtome equipped with a diamond knife (Ultracut E, Reichert-Jung, Heidelberg, Germany) and transferred to copper grids (150 mesh) with a film of polyvinyl butyral (Mowital). The probes were contrasted with solutions of 2% uranyl acetate and 2% lead citrate for 10 min each. Images were acquired at 100kV on a transmission electron microscope (LEO 906E, Zeiss, Jena, Germany) equipped with a CCD camera (MultiScan 794, Gatan, Pleasanton, CA, U.S.A.).
Ultra-thin sections (~80 nm) were prepared on a microtome equipped with a diamond knife (Ultracut E, Reichert-Jung, Heidelberg, Germany) and transferred to copper grids (150 mesh) with a film of polyvinyl butyral (Mowital). The probes were contrasted with solutions of 2% uranyl acetate and 2% lead citrate for 10 min each. Images were acquired at 100kV on a transmission electron microscope (LEO 906E, Zeiss, Jena, Germany) equipped with a CCD camera (MultiScan 794, Gatan, Pleasanton, CA, U.S.A.).
3
Spore Ultrastructure Microscopy Preparation
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Diluted whole spores or purified exosporium (3 μl) were applied to glow discharged carbon-coated Cu/Pd grids; after 1 min the grid was blotted, washed once with (0.75%) uranyl formate and then stained for 20 s. Excess stain was removed by blotting followed by vacuum drying and the grid examined in a Philips CM100 electron microscope operating at 100 kV. Images were recorded on a Gatan MultiScan 794 1 k × 1 k CCD camera at between 3000 and 52,000× magnification and 500–1200 nm underfocus. 10 length measurements from hairy nap, and other filaments were taken and the mean values with standard deviations are shown in the data.
4
Cryo-EM Sample Preparation Protocol for E. coli
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Spore suspension or sonicated E. coli cells (2 μl) were applied to glow-discharged carbon-coated copper palladium grids, incubated for 1 min, and then washed, stained (20 s) with uranyl formate (0.75%), and vacuum dried. For CsxA crystals extracted from sonicated E. coli cells, after sample incubation, the grids were washed once in distilled water before being stained. Grids were examined in a Philips CM100 transmission electron microscope operating at 100 kV. Micrographs were recorded under low-dose conditions on a Gatan MultiScan 794 1,000-pixel-by-1,000-pixel charge-coupled-device (CCD) camera at nominal ×52,000 magnification with 0.5-to-1.2-μm underfocus and at specimen tilts over a range of ±55° in 10° steps.
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