Gatan 626 cryo holder

The consortium was visualized in mid-stationary phase (12 h incubation, MMSY, 0.6 mM SMX) by Cryo-Transmission Electron Microscopy (Cryo-TEM) for morphological characterization. Briefly, a 4 μl aliquot of the overnight grown liquid culture was adsorbed onto a holey carbon-coated grid (Lacey, Tedpella, USA), blotted with Whatman 1 filter paper and vitrified into liquid ethane at − 180 °C using a vitrobot (FEI, USA). Frozen grids were transferred onto a Talos Electron microscope (FEI, USA) using a Gatan 626 cryo-holder (GATAN, USA). Electron micrographs were recorded at an accelerating voltage of 200 kV using a low-dose system (20 to 40 e−/Å2) and keeping the sample at − 175 °C. Defocus values were − 3 to 6 μm. Micrographs were recorded on 4 K × 4 K Ceta CMOS camera. The cell size, and periplasmic and cell wall thickness were measured with Fiji from the ImageJ platform [101 (link)]. For Transmission Electron Microscopy (TEM) analyses, 4 μl aliquot of the sample was adsorbed onto a glow-discharged carbon film-coated copper grid, and subsequently negatively stained with 2% uranyl acetate. Images were recorded using Philips CM200FEG electron microscope operating at 200 kV on TemCam-F416 CMOS camera (TVIPS, Germany).

The Cryo-TEM was conducted following our previously described protocol^{35 (link)}. Briefly, a 3 µL sample of NP in PBS was dispensed onto a glow discharged copper grid (300 mesh) with lacey carbon film coating (ProSciTech, QLD, Australia). The grid was blotted against filter paper (Whatman 541) and plunged into liquid ethane using an in-house plunge freezing device in 80% humidity. The samples were presented to the transmission electron microscope TEM (FEI, Eindhoven, The Netherlands) operated at 120 kV using a Gatan 626 cryo-holder (Gatan, Pleasanton, CA, USA). A low electron dose of 8–10 electrons/Ų was employed. Images were captured using a FEI Eagle 4kx4k CCD camera (FEI) and AnalySIS software v3.2 (Olympus.).

Cryo-TEM images were obtained using a BIO-TEM installed at the Korea Basic Science Institute (Osong, Korea). The isolated ASC-exosomes were applied to Quantifoil grids (Electron Microscopy Sciences, Hatfield, PA, USA) and subsequently blotted using Vitrobot Mark IV (FEI, Hillsboro, OR, USA) with the following settings: 100% humidity, 4 °C, blot time of 5 s, blot force of 5, blot total of 1, wait time of 5 s, and drain time of 0 s. For maintenance of vitrified sample grids at a temperature of around −178 °C within the TEM, a side entry Gatan 626 cryo-holder (Gatan, Pleasanton, CA, USA) was used. The grids were then examined with a Tecnai G2 Spirit Twin TEM equipped with an anti-contaminator (FEI Company, Hillsboro, OR, USA). A 4K × 4K, Ultrascan 4000 CCD camera (Gatan) was used for image recording. A low-dose method (exposures at 1000 electrons per nm²/s) was used for cryo-TEM.

For devitrification experiments, freshly glow discharged C-Flat grids (2/1-2C, Electron Microscopy Science, USA) with deionised water were plunge-frozen using a Leica EM-GP, with blotting time 1 sec. Grids were loaded in the cryoFLM set-up, and illuminated with 488 nm laser light as described, for various intensities and durations (Fig. 1). On a single grid up to 10 different conditions could be examined, leaving at least one grid square between locations to avoid influence from previously illuminated areas. It should be noted however, we did not detect any effect on neighbouring grid squares, even after a grid square was illuminated for several minutes using maximum illumination intensity (83.0 W/cm²). Furthermore, we note that the intensity threshold is expected to be dependent on experimental setup (e.g. support material and mesh size). Grids were then transferred to a Gatan 626 cryo holder (Gatan, Pleasanton, USA) for inspection with cryoEM. The distinction between devitrified and vitreous samples is immediately apparent in the cryoEM, as is shown in Fig. 1.

Stock solutions of beta-amyloid (1–42) and inactive control were resuspended in HEPES 25 mM. A 5 µL drop of each sample was deposited on glow-discharged copper grids (Quantifoil R2/2, Quantifoil Micro Tools), then frozen by rapid immersion in liquid ethane using a Leica EM-GP (Leica Microsystems). Grids were then transferred under liquid nitrogen to a Gatan 626 cryo-holder (Gatan) and examined on a Talos 200C S/TEM (Thermo Scientific, Coon Rapids, MN, USA), equipped with a 4 k × 4 k Ceta camera. Images were acquired using low dose settings, at a nominal magnification of 36,000× and at 3 to 5 µm under focus, giving a final object sampling of 4.1 Å per pixel.