Tecnai spirit tem

For transmission electron microscopy (TEM) analysis, 1 × 10⁸ sperm cells suspended in Tyrode’s albumin lactate pyruvate media [22 (link)] were inoculated with ASFV “ArmeniaΔ285L-GFPhuCD4” [14 (link)], gt II (MOI 5). Sperm cells were incubated with the virus suspension (48 h, 37°C, 5% CO₂, humidified atmosphere). A negative sperm cell culture control was treated similarly to verify appropriate handling and culture conditions.
Subsequently, inoculated sperm cells were washed three times in 1× PBS by centrifugation at 134× g, 10 min, 23 °C. The resulting pellet was treated with fixing solution (2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate (pH 7.2), 300 mosmol, Serva Electrophoresis, Heidelberg, Germany) for at least 2 h at 4 °C, and embedded in 1.8% low-melting agarose (Biozym). Small pieces were postfixed in 1.0% aqueous OsO4, and stained en bloc with uranyl acetate. After stepwise dehydration in ethanol, cells were cleared in propylene oxide, infiltrated with Glycid Ether 100 (Serva Electrophoresis), and polymerized at 60 °C for 3 days. 60–70 nm ultrathin sections were prepared with an ultramicrotome (UC7, Leica Microsystems, Wetzlar, Germany) and collected on EM grids (300 mesh, Plano). Finally, the sections were counterstained with uranyl acetate and lead citrate and analyzed with a Tecnai-Spirit TEM (FEI) at an accelerating voltage of 80 kV.

For TEM analysis, hBM-MSC-sEVs were isolated as described in the “hBM-MSC-sEV isolation” section from a starting volume of 15 mL of CCM. Following hBM-MSC-sEV isolation, the pellet was suspended in 300 μL of filtered PBS which was then filtered using vivaspin 300 kDa filters (Satorius, Cat#VS0651), following a rinse of the filters with 200 μL of filtered PBS centrifuged at 2000×g for 3 min. The concentrated hBM-MSC-sEVs were suspended in an equal volume of 4% PFA for 30 min. Next, two 50 μL drops of hBM-MSC-sEV/PFA suspension were deposited on parafilm and on which carbon-coated electron microscopy grids (Electron Microscopy Sciences, Cat#CF300-CU) were inverted and placed for 5 min on each 50 μL sample followed by a rinse with 50 μL drops of PBS on a sheet of parafilm. Grids were blotted on filter paper to remove excess and let dry for an hour before imaging. The TEM imaging was performed using a FEI Tecnai Spirit TEM with a LaB6 emitter, operating at 120 kV. The images were acquired with an Eagle camera with 4 k × 4 k resolution.

All samples were measured with an FEI Tecnai spirit TEM (FEI, Hillsboro, Oregon, USA) at 120 kV. Images were recorded with a Veleta CCD camera 2048 × 2048 (Olympus-SIS, Münster, Germany) or Eagle CCD camera 4096 × 4096 (FEI, Hillsboro, Oregon, USA) and processed using ImageJ software as described in the supporting information (Section 1 of Additional file 1: Supplementary information).
Single AuNPs were characterized using conventional TEM: Briefly, single AuNPs suspension (10 μL) was deposited onto a 400 mesh carbon-coated copper grid and let dried at room temperature. Images were recorded with the Veleta camera.
Aggregated AuNPs were characterized using cryo-TEM: Aggregated AuNPs suspension (5 μL) was deposited on a carbon-coated copper grid and liquid excess was carefully removed with filter paper. The grid was then plunged into a liquid ethane bath cooled by liquid nitrogen. The resulting vitrified sample was then stored in liquid nitrogen prior to analysis. Images were recorded using the Eagle camera.