Jem 3100fef

Grids were transferred into a transmission electron microscope (JEM-3100FEF; JEOL) with a high-tilt liquid nitrogen cryotransfer holder (914; Gatan, Inc.). Tilt series images were recorded at −180°C using a 4,096 × 4,096–pixel complementary metal-oxide semiconductor camera (TemCam-F416; Tietz Video and Image Processing Systems) and automated acquisition using Recorder software (System in Frontier, Inc.). The angular range of the tilt series was from −65 to 65° with 2.0° increments. The total electron dose was limited to ∼100 e⁻/Ų. Images were recorded at 300 keV, with 6–12-µm defocus, at a magnification of 25,700× and a pixel size of 6 Å. An in-column Ω energy filter was used to enhance image contrast in the zero-loss mode with a slit width of 23 eV.

The exosomal fraction isolated from pFF was resuspended in NaHCa buffer (30 mM HEPES, 100 mM NaCl, 2 mM CaCl₂, pH 7.4). The resuspended exosomal
fraction was applied on a 200-mesh copper microgrid covered with a formvar support film, which had been pre-treated with soft plasma etching equipment (SEDE-AF,
Meiwafosis, Tokyo, Japan) to obtain a hydrophilic surface. The microgrid was electron-stained with 1% uranium acetate solution for 10 min and then with lead
acetate solution for 10 min. Finally, the microgrid was observed with a transmission electron microscope (JEM-1010; JEOL, Tokyo, Japan).
For cryogenic TEM (Cryo-TEM), the resuspended exosomal fraction in NaHCa buffer was applied on a 200-mesh copper microgrid that had been pre-treated with a
glow-discharger (HDT-400, JEOL) to obtain a hydrophilic surface. The microgrid was immediately plunged into liquid propane using a specimen preparation machine
(EM CPC; Leica) and then transferred to a cryo-transfer holder (Model 626-DH; Gatan, Pleasanton, CA, USA). Finally, the microgrid was observed with a
transmission electron microscope (JEM-3100FEF; JEOL) at an acceleration voltage of 300 kV in zero-loss imaging mode. The temperature of the microgrid was
maintained below –140°C during the observation.

Grids were transferred to a JEM-3100FEF or JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan) with a Gatan 914 (3100FEF) or 626 (2100F) high-tilt liquid nitrogen cryotransfer holder (Gatan, Pleasanton, CA). Tilt series images were recorded at -180°C using Gatan K2 summit direct detector (3100FEF) or a TemCam-F216 CMOS camera (2100F; TVIPS, Gauting, Germany) and automated acquisition was performed using SerialEM software (3100FEF) (Mastronarde, 2005 (link)) or EM-TOOLs program (2100F; TVIPS). The angular range of the tilt series was from -60° to 60° (3100FEF) or -45° to 45° (2100F) with 2.0° increments. The total electron dose was limited to ∼100 e^–/Ų. Images were recorded at 300 keV, with 6–9 μm defocus (3100FEF) or 200 keV with 3- to 5-μm defocus (2100F), at a magnification of ×7100 and a pixel size of 7 Å (3100FEF) or ×19,500 and a pixel size of 8 Å (2100F). With JEM-3100FEF, an in-column omega energy filter was used to enhance image contrast in the zero-loss mode with a slit width of 40 eV.

Thin-section electron microscopy of the axonemes was performed as previously described (Huang et al., 1979 (link)). The cacodylate buffer in the method was substituted by 50 mM Na-phosphate, pH 7.0. The samples were observed by a JEM-3100FEF transmission electron microscope (JEOL, Tokyo, Japan) equipped with TemCam-F416 (TVIPS, Gauting, Germany). Images were recorded at 300 keV, with ∼3-μm defocus, at a magnification of ×82,100 and a pixel size of 1.9 Å. Cross-sectional images in which the PFs were clearly visible were selected and used for averaging of DMTs. Alignment and averaging of DMTs were conducted using custom Ruby-Helix scripts (Metlagel et al., 2007 (link)) and the align2d program of EMAN (Ludtke et al., 1999 (link)).