Sigma scanning electron microscope

Transmission electron microscopy (TEM) was performed on a JEOL electron microscope at an accelerating voltage of 100 kV. High-angle annular dark-field scanning TEM (HAADF-STEM), HAADF-STEM energy dispersive X-ray spectroscopy (HAADF-STEM-EDS), and high-resolution TEM (HRTEM) were performed on an FEI Tecnai F30 transmission electron microscope at an accelerating voltage of 300 kV. Scanning electron microscopy EDS (SEM-EDS) was conducted on a ZEISS Sigma scanning electron microscope at an accelerating voltage of 20 kV. Inductively coupled plasma optical spectroscopy (ICP-OES) was carried out on a Thermo Fisher iCAP700 series (Thermo Fisher Scientific, Waltham, MA USA) in axial mode. X-ray diffraction spectroscopy (XRD) was conducted on a Rigaku XRD machine with Cu Kα (λ = 1.540598 Å). X-ray photoelectron spectroscopy (XPS) was conducted on an SSI S-Probe XPS spectrometer. The XANES and EXAFS spectra were measured in transmission mode by using the TPS 44A beamline at the National Synchrotron Radiation Research Centre (NSRRC, Hsinchu).

These data (Fig. 2 and Supplementary Fig. 1) were collected via energy-dispersive spectroscopy on a Carl Zeiss Sigma scanning electron microscope at the University of Glasgow. Samples were carbon-coated, and the electron microscope was operated in high-vacuum mode at 20 kV and ~ 2 nA.

For electron microscopy, a small piece of mouse renal cortex was immersion fixed in 4% PFA and 1% (v/v) glutaraldehyde in 0.1 M PB. After post-fixation (same fixative o/n at 4 °C), tissue blocks were washed in 0.1 M PB, treated with OsO₄ (0.5% for 60 min), and stained with uranyl acetate (1% w/v in 70% v/v ethanol). After dehydration, tissue blocks were embedded in Araldite resin (Serva Electrophoresis GmbH, Heidelberg, Germany). Eighty nanometer ultrathin sections were cut on an UC6 ultramicrotome (Leica, Wetzlar, Germany), rinsed in lead citrate buffer before analysis using a Zeiss Sigma scanning electron microscope using a STEM detector (Zeiss, Oberkochen, Germany).

The resin-embedded sections were cut into 30 nm serial ultrathin sections using automated tape-collecting ultramicrotome (ATUM) ^{86 (link)}. Serial sections were collected onto carbon-coated and plasma-treated Kapton tape. The tape was cut into strips and affixed onto 150 mm silicon wafers (University Wafer).
A Zeiss Sigma scanning electron microscope was used to acquire overview images from the serial sections. Two overview images were taken per wafer, which were around 1.35 μm apart in z-axis. Typical imaging conditions are 8-kV landing energy, 1.2-nA beam current, 3-μs dwell time, 150-nm pixel size, and 4k × 4k images. The images were captured using a below-the-lens backscatter detector and Zeiss’ Atlas 5 software. The overview images were aligned using the “Linear stack alignment with SIFT” plugin in FIJI.
Prior to acquiring high-resolution images, the serial section sections on wafers were post-stained for 4 min with a 3% lead citrate solution. After staining, the sections were degassed for a minimum of 24 h at 1×10–6 Torr. A Zeiss MultiSEM 505 scanning electron microscope equipped with 61 electron beams was used to acquire high-resolution images from the serial sections. Images were collected using a 1.5-kV landing energy, 4-nm image pixel, and a 400-ns dwell time.

After micro-CT imaging, we bisected a brain sample with a bandsaw and machined a 7 mm × 7 mm × 20 mm block of cortex from one hemisphere. We mounted the block onto the chuck of a Leica EM UC-7 Ultramicrotome (Leica Microsystems, Buffalo Grove, IL), trimmed down the machining damage with a diamond knife and reduced the face to a 1 mm × 2 mm rectangle with a 400 μm setback. We then collected 30 nm sections onto carbon-coated Kapton tape with an automated tape-collecting ultramicrotome (ATUM)⁴⁹ . The tape with the sections was mounted to silicon wafers and we then post-stained the sections with 1% uranyl acetate in maleate buffer and 3% stabilized lead citrate (Ultrostain II, Leica Biosystems, Wetzlar, Germany). We imaged the sections on a Zeiss Sigma scanning electron microscope and acquired images using secondary electron detection.