914 cryo holder

An 8 μl aliquot of sample was pipetted onto a carbon coated copper grid (HC300Cu, Holey Carbon film on Copper 300 mesh, EM Resolutions, UK). Excess liquid was blotted away with filter paper (Whatman number 1) and the grid was plunge-frozen in a liquid mixture of ethane/propane cooled by liquid nitrogen. The sample was then kept at liquid nitrogen temperatures throughout the analysis. TEM images were taken on a JEOL 2200FS TEM at 200 keV using a Gatan K2 Summit and Gatan 914 cryo-holder.

The hydrodynamic diameter and size distribution of the NCs were measured with a cryogenic transmission electron microscope (cryo-TEM). Aqueous NC dispersions (3 μL) were loaded on a glow discharged (EmiTech K100 machine, Quorum Technologies, East Sussex, UK) Quantifoil or lacey grid, which was blotted and plunged into liquid ethane using a Gatan CP3 automated plunger (Gatan, Pleasanton, CA, USA) and stored in liquid nitrogen until use. Frozen specimens (samples with vesicles embedded in vitreous ice) were transferred to a Gatan 914 cryo-holder and maintained at temperatures below −176 °C inside the microscope. Samples were inspected with a FEI Tecnai G2 microscope (now Thermo-Fisher Scientific, Waltham, MA, USA) with an acceleration voltage of 120 kV equipped with a cryobox decontaminator. Images were taken using a digital micrograph (Gatan, ver. 1.83.842) with a mulitiscan camera (Gatan, model 794) at varying resolution. The mean diameter was determined by measuring at least 200 particles using the image analysis software AnalySIS Auto version 3.2 (Soft Imaging System GmbH, Münster, Germany). Each experiment was repeated at least three times.

Initial tomography experiments were performed on a JEOL 2200 FS equipped with a Gatan Ultrascan 4000 CCD detector and 914 cryo‐holder. Continuous tilt‐series were collected, using SerialEM, from −60° to +60° at 2° intervals and at a nominal magnification of 20k×, corresponding to an unbinned pixel size of 5.32 Å at the specimen scale. Improved tomography data, suitable for sub‐tomogram averaging, were then collected at the UK electron bio‐imaging centre at Diamond Light Source (eBIC) on a Thermo Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered direct detection camera. Dose‐symmetric tilt‐series collection was performed using SerialEM (Mastronarde, 2005 (link); Hagen et al, 2017 (link)). Energy‐filtered images were collected with a slit‐width of 20eV and an applied defocus of between −2 and −4.5 μm. One‐second exposures were recorded with an electron exposure of 2 electrons/Ų, partitioned over five movie frames. A total of 41 images were recorded per tilt‐series at 3° intervals from −60° to +60°, and thus, a total exposure of 82 electrons/Ų was applied per tilt‐series. Tilt‐series were recorded at a nominal column magnification of 81k× corresponding to a calibrated pixel size of 1.79 Å at the specimen scale.