Holey carbon film grid

Cryoelectron microscopy was performed as described previously [28 (link)]. Briefly, nanoparticles were analyzed
by spotting 3 µL of the specimen to a holey carbon-film grid (Quantifoil Micro
Tools GmbH, Jena, Germany) pretreated with Gatan Solarus 950 plasma cleaner. The
grid was shock-frozen in liquid ethane using a Gatan Cryoplunge 3 (Gatan Inc,
Pleasanton, CA, USA). Low dose imaging of the frozen, hydrated specimen kept at
liquid nitrogen temperature with a Gatan 626 single tilt cryoholder was
performed in a JEM2100 electron microscope (JEOL Ltd, Tokyo, Japan) operating at
200 kV. All images were recorded at ~40,000 x magnification using a Gatan
UltraScan 4000 CCD camera.

Liposomes were prepared as described and analyzed by cryo-electron microscopy as previously described in [14 (link)]. Briefly, 1 mL plasmid DNA-loaded liposomes were applied over a holey carbon-film grid (QuantifoilMicro Tools, Jena, Germany), followed by flash-freezing in liquid ethane using a Gatan Cryoplunge 3 (Gatan, Pleasanton, CA, USA). The visualization of the frozen, hydrated specimen was performed on a JEM2100 electron microscope (JEOL, Tokyo, Japan, operating at 200 kV) resulting in images of 2 μm scale. Image recording was done with a Gatan Ultrascan 4000 CCD camera at 40,000× magnification.

Liposomes were prepared as described above and analyzed by cryoelectron microscopy as previously published.⁵¹ (link) For this, 3 μL DNA-loaded liposomes were spotted to a holey carbon-film grid (Quantifoil Micro Tools, Jena, Germany), previously pretreated with Gatan Solarus 950 plasma cleaner. Specimen-coated grids were plunge-frozen in liquid ethane using a Gatan Cryoplunge 3 (Gatan, Pleasanton, CA, USA). Low-dose imaging of the frozen, hydrated specimen kept in liquid nitrogen with a Gatan 626 single tilt cryoholder was performed on a JEM2100 electron microscope (JEOL, Tokyo, Japan, operating at 200 kV). A Gatan Ultrascan 4000 CCD camera was used to record images (∼40,000× magnification).