Tecnai f20 transmission electron microscope

The adipose tissues were dissected and fixed with 2.5% (vol/vol) glutaraldehyde in 0.2 M cacodylate buffer overnight. After washing in 0.2 M PBS, the tissues were cut into small pieces of approximately 1 mm³ and immersed in 1% OsO₄ in 0.2 M cacodylate buffer for 2 h at 4°C. Then, the samples were dehydrated through a graded ethanol series and embedded in resin (Low Viscosity Embedding Media Spurr's Kit, EMS, 14300). ADSCs were fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) overnight at 4°C. After being washed with PBS, the samples were post-fixed with 1% osmium tetroxide containing 0.8% potassium ferricyanide at room temperature for 1 h, embedded in Spurr's resin. Ultrathin sections were cut on an ultramicrotome, stained with uranyl acetate and lead citrate, and observed using FEI Tecnai F20 transmission electron microscope (Oregon, USA).

UV–Vis spectroscopy was performed using a Cary 60 UV–Vis spectrometer (Agilent Technologies, Santa Clara, CA, USA). Transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) mapping were performed using an FEI Tecnai F20 transmission electron microscope (FEI Company, Eindhoven, the Netherlands). The hydrodynamic size of the nanoparticles was measured using ζ-potentials (ELS-Z, Otsuka, Japan). Fourier transform infrared (FT-IR) spectra were recorded on a PerkinElmer spectrometer in the range between 4000 and 400 cm⁻¹.

Cryo-ET data were collected with single-axis tilt using either a Tecnai F20 transmission electron microscope (TF20, FEI) equipped with an Eagle 4K×4K multiport CCD camera (FEI), or a Titan Krios (FEI) with a K2 Summit direct electron detector (K2 camera, Gatan). The TF20 was operated at an acceleration voltage of 200 KV, with tilt series collected from −60° to +60° at 2° intervals using FEI Xplore 3D software. Defocus values of −12 to −18 μm and total electron dosage of ~100 e⁻/Ų were used for all imaging with TF20, with final pixel size of 0.755 nm. The Titan Krios was operated at an acceleration voltage of 300 KV. Images were collected by the K2 camera in counting mode, with tilt series acquired from −64° to +64° at 2° intervals using Leginon (Suloway et al., 2005 (link)). A defocus value of −10 μm and total accumulated dose of ~120 e⁻/Ų was used for all imaging with Titan Krios, with final pixel size of 0.765 nm.

Purified wild-type or fcpB⁻ flagella were applied to glow-discharged Quantifoil holey carbon grids, blotted and plunge-frozen using a Vitrobot Mark III (FEI Company, Eindhoven, The Netherlands). Cryo-EM images were collected using a Tecnai F20 transmission electron microscope (FEI Company) operating at 200 kV. Micrographs were acquired at ~52 KX magnification in SerialEM (Mastronarde, 2005 (link)) using an UltraScan 4000 (4 K by 4 K) CCD camera (Gatan, Inc., Pleasanton, CA, USA) at the Yale School of Medicine, Center for Cellular and Molecular Imaging. Box segments of imaged filaments were manually selected using the boxer program from the EMAN software suite (Tang et al., 2007 (link)). CTF-correction was carried out on the selected particles using Gctf (Zhang, 2016 (link)) and then subjected to iterative reference-free 2D classification and alignment using RELION (Scheres, 2012 (link)), with N = 50 classes. Following this step, a subset of class averages with well-resolved features were identified for both wild-type (29) and fcpB⁻ mutant (13) samples, and in each case the majority of class averages (~19/29 WT; ~8/13 fcpB⁻ mutant) were nearly indistinguishable from the examples shown in Figure 6E. The highly limited number of distinct views of the flagellum derived from 2D classification is likely due to the constrained orientation of the curved filament within the ice layer.

To characterise the particle size distribution and morphology of the iron oxide nanoparticles, very small droplets of the individual FeO_xNP dispersions and the mixture were pipetted onto TEM grids and the excess moisture was allowed to dry. To measure the size of the primary particles, TEM imaging of samples was carried out using a FEI Tecnai F20 Transmission Electron Microscope working on high tension at 200 kV, with an extraction voltage of 4450 eV. Bright-field particle imaging in TEM mode was carried out using the Gatan Digital Micrograph attached to the instrument. For identifying likely particles, the instrument scanning TEM (STEM) mode was used in conjunction with high angle annular dark field (HAADF) detection. The Gatan Digital Micrograph software was used to measure the particle diameters of 40 NPs of each iron oxide. Additional high-resolution TEM images were taken with a JEOL 3000F Transmission Electron Microscope (JEOL Inc., Tokyo, Japan).