T12 microscope

A Ni²⁺-NTA lipid-containing monolayer system was used to reconstitute the chemotaxis core-signaling complex arrays, as described previously¹⁶ . A mixture of 9:18:18 µM of TarCF:CheA:CheW in a buffer containing 75 mM Tris-HCl, pH 7.4, 100 mM KCl, and 5 mM MgCl₂ was applied to a Teflon well, over which we immediately laid a lipid monolayer containing 2:1 DOPC:DOGS-NTA-Ni²⁺ lipid mixture, at 2 mg mL⁻¹ concentration. The monolayer set up was left undisturbed in a humidity chamber overnight. The monolayer specimen was picked up with holey carbon grids, stained with 1% uranyl acetate, and examined with an FEI T12 microscope operated at 120KV.

Scanning electron microscopy images were attained by the staff at the Electron Microscopy Resource Laboratory (EMRL) at the University of Pennsylvania. In brief, aggregates were washed, fixed in glutaraldehyde, dehydrated, gold sputter coated and subsequently imaged with a scanning electron microscope at 3000x with a FEI-Tecnai T12 microscope at the PennMed Imaging Core.

Samples (concentrations close to 0.05 mg·mL⁻¹) were applied between a carbon and a mica layer. The carbon was then floated on the top of a heavy atom salt drop (2% (w/v) sodium silicotungstate, pH 7.0). The carbon film was covered by a copper grid. Both were fished using a small piece of paper and air dried before insertion in the electron microscope [43 ,44 (link)]. Charge-coupled Device (CCD) frames were taken with a T12 microscope (FEI, Hillsboro, OR, USA) operating at 120 kV and a nominal magnification of 45,000 times. The ^5′phosphate-polyUC-FAM^3′-NP complex was incubated overnight at room temperature using 100 µM of proteins in 20 mM Tris-HCl pH 7.5, 50, 150 or 300 mM NaCl and 5 mM β-ME and with a final ratio NP:RNA of 1:1. The dilutions for EM were performed with a buffer with the same salt concentration.

To examine the filament before cryo imaging, all flagellar filament samples were negatively stained. The filament samples (3.5 μl) were deposited for 30 s on a glow-discharged 300 mesh carbon-coated cooper grid (Electron Microscopy Sciences). The grid was then washed with 3 drops of 2% uranyl acetate. Images were taken with a Gatan CCD camera on a T12 microscope (FEI) operating at 80 keV. The images of negatively stained wild-type P. aeruginosa filaments were recorded with a 4k × 4k Gatan CCD camera on an F20 microscope (FEI) operating at 200 keV, in order to generate a power spectrum for analysis.

An aliquot of 4 μl Piezo1 (0.05 mg/ml) was applied to glow-discharged carbon-coated copper grids (200 mesh, Zhongjingkeyi, Beijing). After the grids were incubated at room temperature for 1 min, excessive liquid was absorbed by filter paper. Grids containing the specimen were stained by applying droplets of 2% uranyl acetate for 30 s and then air-dried. Micrographs were taken on a T12 microscope (FEI) operated at 120 kV, using a 4k × 4k CCD camera (UltraScan 4000, Gatan). Images of Piezo1 at pH 7.4 and pH 5.0 were taken at a nominal magnification of 49 000 times.