Tecnai g2

For negative staining analysis, membrane proteins were extracted with 5% SMA and nAChRs were enriched using α-Btx affinity pull-downs. Proteins were diluted 1:10 with deionised water to approximately 0.9 mg/ml and an aliquot of the samples were absorbed onto a glow-discharged copper/carbon-film grid (EM Resolutions) for approximately 2 min at room temperature. Grids were rinsed twice in deionised water and negative staining was performed using a 2% aqueous uranyl acetate solution. Samples were viewed in a Tecnai G2 transmission electron microscope (TEM, FEI/ThermoFisher) run at 200 keV accelerating voltage using a 20 μm objective aperture to increase contrast; images were captured using an AMT CCD camera. Three biological replicates were performed and we provide 28 micrographs, 15 enriched from α-Btx and 13 unenriched (Figure 3—source data 1).

MSC-EVs were fixed with 4% PFA for 5 min, and one drop (2.00 × 10⁹ particles) of EVs was placed on a 400-mesh holey-film grid for 10 min. After washing with PBS, the MSC-EVs were stained with 1% uranyl acetate for 2 min. The sample was then washed with PBS and finally observed with a Tecnai G2 (FEI; Thermo Fisher Scientific, Waltham, MA, USA) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System; Münster, Germany) digital camera.

Lacey carbon 300 mesh grids (Ted Pella, USA) were used in all cryo-EM experiments. We found that lacey networks with holes ranging from 20 nm to few microns entrapped different reconstituted material more than calibrated Quantifoil grids. In all experiments, blotting was carried out on the opposite side from the liquid drop and plunge frozen in liquid ethane (EMGP, Leica, Germany). Samples were imaged using different electron microscopes. Data and 2D images depicted in Fig. 1c, Supplementary Fig. 1b–f, Supplementary Fig. 2a–e, Fig. 3c and Supplementary Fig. 3d were acquired with a Tecnai G2 (Thermofisher, USA) Lab6 microscope operated at 200 kV and equipped with a 4k × 4k CMOS camera (F416, TVIPS). Image acquisition was performed under low dose conditions of 10 e⁻/Ų at a magnification of 50,000 or 29,500 with a pixel size of 2.1 or 3. 6 Å, respectively. Plots depicted in Figs. 1e, 3d were derived from images taken with the Tecnai G2 electron microscope. Data and 2D images depicted in Figs. 1e, f, 2b, plot Fig. 2b were acquired with a Polara (Thermofisher, USA) FEG 300 kV microscope with a K2 Gatan camera in counting mode with 40 frames during 6 sec, total dose 80 e⁻ and at 0.96 Å/px.

The Fe-Si-NP were purchased from Kisker Biotech GmbH, Steinfurt, Germany (order No. PMSI-H.25–5). According to manufacturer, particles are composed of a maghemite Fe₂O₃ core and an amorphous SiO₂ shell and had a surface area of 50 m²/g, according to the Brunauer–Emmet–Teller (BET) method. To further characterize particle size and shape by transmission electron microscopy (TEM), we dried 0.5 µL of the aqueous suspension, as used for intratracheal instillation (6 mg/mL H₂O), onto carbon-coated copper grids. TEM analysis was carried out with a Tecnai G2 (ThermoFisher Scientific, Waltham, MA, USA). The size distribution of these aggregates (in H₂O) was measured with a NanoSight LM10 instrument equipped with a green laser (532 nm), an Andor CCD camera, and NTA software 2.1 (Malvern Instruments GmbH, Herrenberg, Germany).

The coenzyme F420 contains a fluorescent component [30 ], and the oxidation states of the coenzyme F420 can absorb 420 nm ultraviolet light and excite 470 nm blue-green fluorescence [30 ]. Isolated cells from pure cultures were observed under a fluorescence microscope (BX63 Olympus, Tokyo, Japan) with 405 nm ultraviolet light as the light source. For observation under electron microscopes, we centrifuged the cell suspension at 6000× g for one minute and washed it three times with 0.5 mL sterile water. We then fixed the cells on a silicon pellet illuminated by a white light source and observed them under a scanning electron microscope (VEGA 3, TESCAN, Brno-Kohoutovice, Czech Republic). Alternatively, the cells were fixed on copper grids and observed under a transmission electron microscope (Tecnai G2, Thermo Fisher Scientific, Waltham, MA, USA).