Digital micrograph

The immunogold protocol was described in our previous study (60 (link)). Briefly, E. coli WT and GM1 were grown as described above, and the optical density at 600 nm (OD₆₀₀) was measured and adjusted to 1.0. Next, 2 ml of the cells was incubated with 2 μl of CTB (1 mg ml⁻¹; Sigma) for 1 h before being washed and resuspended in PBS. The cell suspension was spotted onto Parafilm and a Formvar-coated copper grid laid atop for 1 h. The grid was then blocked with PBS plus 5% bovine serum albumin (BSA) for 1 h and treated with rabbit α-CT antibodies (Fitzgerald Industries International) in PBS plus 5% BSA for 1 h and then goat α-rabbit IgG conjugated to 10-nm gold particles (BB International) in PBS plus 5% BSA. Both antibodies were used at a 1:50 dilution, and grids were washed in PBS plus 5% BSA three times between each step. The grids were imaged using a Philips Morgagni 268 transmission electron microscope (FEI Company) along with a charge-coupled camera and controller (Gatan). The images were processed using DigitalMicrograph (Gatan).

For fluorescence microscopy, C. albicans cells were fixed in 50 mM thimerosal and stained for exposed β-glucan (1.5 µg/ml Fc-Dectin-1 plus anti-human IgG conjugated to Alexafluor 488; green) and exposed mannan (25 µg/ml Concanavalin A conjugated to Alexafluor 647; red). All samples were examined by phase differential interference contrast (DIC) and fluorescence microscopy using a Zeiss Axioplan 2 microscope. Images were recorded digitally using Openlab v 4.04 (Improvision, Coventry, UK) with a Hamamatsu C4742- 95 digital camera (Hamamatsu Photonics, Japan). Fluorescence was quantified using and processed using Openlab (Openlab v 4.04: Improvision, Coventry, UK).
C. albicans cell walls were examined by high-pressure freeze substitution transmission electron microscopy (TEM). C. albicans SC5314 cells were incubated for 6 h at 37 °C with extracts from the small intestine, cecum or large intestine (above). These fungal cells were then processed for TEM as described previously (Ene et al., 2012 (link)), cutting ultrathin (100 nm) sections. Samples were imaged with a Philips CM10 transmission microscope (FEI, United Kingdom) equipped with a Gatan Bioscan 792 camera, and the images recorded using a Digital Micrograph (Gatan, Abingdon Oxon, United Kingdom).

3 μl of proteins (~0.05 mg/ml) diluted with PBS buffer were deposited onto carbon-coated grids (EMS CF200-Cu) glow-discharged for 15 seconds at 25 mA in air. After 60 s incubation, excess proteins were wicked by filter paper. Grids were washed once in buffer, and stained twice in Nano-W stain (Nanoprobes) with blotting in between. The grids were air dried on filter paper and imaged using a Tecnai T12 Spirit operated at 120 kV. Micrographs were taken in Digital Micrograph (Gatan).

Transmission electron microscopy was performed with two microscopes (JEOL JEM-F200 and an aberration-corrected JEOL NEOARM), both operating at 200 kV and in scanning transmission electron microscopy (STEM) mode. For ex situ studies, the sample was diluted in isopropanol and deposited on a lacey carbon film on copper grids. For in situ studies, an environmental holder for gas flow experiments manufactured by Hummingbird Scientific was used. The sample was enclosed in a microchip, made of two SiN windows and a micro electro-mechanical system for temperature control. The mass flow of gases was controlled using a gas system and a software provided by the same company.
Energy-dispersive X-ray spectroscopy (EDS) was performed with JEOL NEOARM and the maps were obtained with DigitalMicrograph by Gatan Inc (pixel time = 0.04 s and pixel size = 1.4 Å).

The strain fields in this article were deduced for all HAADF-STEM images using custom plugins of GPA for Gatan Digital Micrograph^{46 (link),47 (link)}. The strain in the STO lattice is relatively smaller and the STO layer is usually used as a reference. The lattice parameters of PTO and STO are slightly different, and a normalization process has been carried out. The GPA is an effective approach to determine crystal lattice variations over a larger area. To further prove that the GPA results are credible and show correspondence with the STEM images, we calculated the a/c ratio and compared it with the GPA results as shown in Supplementary Fig. 7; they show a high degree of consistency.

The rats were sacrificed and perfused overnight in Karnovsky's fixative. The brain samples were fixed in 2.5% glutaraldehyde for 2 h and sectioned as described previously 67 (link). TEM images were captured using a high-resolution charge-coupled device (CCD) camera. Images were processed using a digital micrograph (Gatan, USA). G-ratios were analyzed using Image J software (NIH, USA), and ~100 remyelinated axons were measured for each group.

Average size, distribution and morphology were analysed by transmission electron microscopy (TEM) using a FEI Tecnai T20 microscope, operated at 200 keV. The average particle size () and size distribution was calculated from histograms after counting N > 500 particles of both Fe₃O₄ cores and Au NPs. The data could be fitted with a lognormal distribution. High resolution transmission electron microscopy (HRTEM images were taken using a FEI Tecnai F30 microscope, operated at an acceleration voltage of 300 KV. The microscope was equipped with a HAADF (high angle annular dark field) detector for the STEM mode and EDX (X-ray energy disperse spectrometry) pattern was also studied. Lattice fringes were measured from the fast-Fourier transform of HRTEM images, using Gatan Digital Micrograph. Samples of NPs were prepared by placing one drop of a dilute suspension of NPs in ethanol on a carbon-coated copper grid and evaporating the solvent at room temperature. HRTEM images were used for studied the morphology, grain size and structural information of our samples.