Emmanu4 v

Vesicles isolated from 5 × 10⁴ infected monocytes (MOI 1:1) were collected in 5-µL suspension and enclosed between two 0.1-mm-thick copper profiles as used for the freeze-fracture sandwich double-replica technique. The sandwiches were physically fixed by rapid plunge-freezing in a liquid ethane/propane mixture, cooled by liquid nitrogen. Freeze-fracturing was performed at −150 °C in a BAF400T freeze-fracture unit (BAL-TEC) using a double-replica stage. The fractured samples were shadowed with 2 nm Pt/C (platinum/carbon) at an angle of 35 °C, followed by perpendicular evaporation of a 15–20-nm-thick carbon layer. The evaporation of Pt/C was controlled by a thin-layer quartz crystal monitor; the thickness of the carbon layer was controlled optically. The obtained freeze-fracture replica was transferred to a cleaning solution (commercial sodium hypochlorite, containing 12% active Cl₂) for 30 min at 45 °C. Then, the replica was washed four times in distilled water and transferred onto unfiled copper EM-grids for examination in a Zeiss EM902A electron microscope (Carl Zeiss) operated at 80 kV. Images were recorded with a 1 k (1024 × 1024) FastScan-CCD-camera (CCD-camera and acquisition software EMMANU4 v 4.00.9.17, TVIPS).

EVs isolated from 4 × 10⁴ infected monocytes (MOI 1:1) were collected in 4-µL suspension, and were applied onto copper EM-grids covered by a QUANTIFOIL Multi A holey carbon film (Quantifoil Micro Tools), and excess of liquid was blotted automatically between two strips of filter paper. Subsequently, the samples were rapidly plunge-frozen in liquid ethane (cooled by liquid nitrogen at about −180 °C) in a cryobox (Carl Zeiss). Excess of ethane was removed with a piece of filter paper. The samples were transferred immediately using a Gatan 626 cryo-transfer holder (Gatan) into a pre-cooled cryo-electron microscope (Philips) operated at 120 kV and imaged under low dose conditions. The images were recorded with a 2k F216-CMOS-camera (CMOS-camera and acquisition software, EMMANU4 v 4.00.9.17, TVIPS). In order to minimize the noise, four images were recorded and averaged to one image.