Glow discharged holey carbon grid

For cryo-EM, niosome samples were directly adsorbed onto glow-discharged holey carbon grids (QUANTIFOIL). For vitrification, grids were blotted at 95% humidity and rapidly plunged into liquid ethane with VITROBOT (Maastricht Instruments BV).
For immuno-EM, samples were fixed in 2% paraformaldehyde in PBS and deposited onto Formvar/carbon-coated EM grids, previously pre-coated with Poly-L-lysine (MW of 15,000–30,000 kDa, Sigma Aldrich) as described before [19]. The niosome-coated grids were washed with PBS and finally blocked with PBS/1% BSA for 15 min. Blocked grids were transferred to a drop of PBS/0.1% BSA containing primary antibody and incubated ON at 4°C in a humidified chamber. The grids were washed on a drop of PBS, transferred to a drop of PBS/0.1% BSA containing secondary antibodies conjugated to 15 nm gold-particles (Aurion) and incubated for 2 h at RT in a humidified chamber. Grids were washed with dH₂O and fixed with 1% glutaraldehyde, washed with H₂O and stained with 2% uranyl acetate for 1 min at RT. Afterwards, samples were vitrified as described above and visualised using a JEM-2200FS Field Emission TEM equipped with a digital camera (JEOL).

Nanoparticle preparations were plunge-frozen onto glow discharged holey carbon grids (Quantifoil Micro Tools, Jena, Germany) in liquid ethane using the environment-controlled Vitrobot (Vitrobot MarkIV, FEI, Eindhoven, The Netherlands). Briefly, 3 μl of the nanoparticle solution were applied onto the grid which was held by tweezers inside the climate chamber (22°C, 100% relative humidity) of the Vitrobot. The solution was automatically blotted with filter paper leaving a thin film of the nanoparticle solution over the holes. The film was allowed to relax for 10 s prior to plunge freezing the sample on the grid in liquid ethane cooled near to its freezing point. Vitrified nanoparticle samples were imaged at −170°C using a Gatan cryo-transfer holder (Gatan 626, Gatan Inc. Pleasanton, USA) and standard low-dose imaging conditions (1 e⁻/Ų.s)64 (link) at a Tecnai G2 F20 transmission electron microscope (FEI, Oregon, USA), operated at 200 kV. Images were acquired at × 25,000 magnification on a 2k × 2k CCD camera (894 Ultrascan 1000, Gatan Inc., Pleasanton, USA).