Tecnai 12 twin transmission electron microscope

TEM and Cryo-TEM images were captured on an FEI Tecnai 12 TWIN Transmission Electron Microscope, operating at 100 kV for TEM and 80 kV for cryo-TEM. 25 μM TEM samples were pipetted onto a carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA). Filter paper was used to wick away excess solution. 10uL of 2 wt% aqueous uranyl acetate was used to stain samples. For cryo-TEM, 25 μM samples were pipetted onto lacey carbon coated TEM grids (LC325-Cu, Electron Microscopy Sciences) pre-treated with plasma air to make the lacey carbon film hydrophilic. Samples were vitrified by plunging them into a liquid ethane reservoir precooled with liquid nitrogen. Both TEM and Cryo-TEM images were acquired with a 16 bit 2k × 2k FEI Eagle bottom mount camera (Hillsboro, OR). Images were processed with ImageJ (NIH, Bethesda, MD). Nanoparticle size was averages from three areas of view with more than 50 particles per image for TEM and 20 particles for Cryo-TEM. Bilayer thickness was averaged across three points along the circumference of the vesicle. Numbers are presented as averages ± 95% confidence interval.

For cryo-TEM, 5 μM Nanoworm suspended in dPBS were evaluated. Lacey carbon film copper grids (300 mesh, Electron Microscopy Services, Hatfield, PA, USA) were pretreated with plasma air for 30 s to render the lacey carbon film hydrophilic. A 6 μL sample was applied on the grid using a Vitrobot (FEI, Hillsboro, OR, USA) that was maintained at 95% humidity. Following 1 min incubation, blotting was performed using Vitrobot preset parameters and the grid was immediately plunged into a liquid ethane reservoir precooled by liquid nitrogen. Grids were then transferred to a cryo-holder and cryo-transfer stage that were precooled with liquid nitrogen. A FEI Tecnai 12 Twin Transmission Electron Microscope, operating at 100 kV, was used to perform all imaging. The cryo-holder was maintained below −170 °C with liquid nitrogen to prevent the sublimation of vitreous water during the imaging process. All images were recorded with a 16-bit 2 K × 2 K FEI Eagle bottom mount camera (Hillsboro, OR, USA). The length and the width of Nanoworms were measured using ImageJ (v2.0.0, NIH, MD) based on the reference length presented in Fig. 1D.

6 μL of sample solution was placed on a holey carbon film supported on a TEM copper grid (Electron Microscopy Services, Hatfield, PA, USA). All the TEM grids used for cryo-TEM imaging were treated with plasma air to render the lacey carbon film hydrophilic. A thin film of the sample solution was produced using a Vitrobot with controlled humidity chamber (FEI). After loading of the sample solution, the lacey carbon grid was blotted using preset parameters and plunged instantly into a liquid ethane reservoir pre-cooled by liquid nitrogen. The vitrified samples were then transferred to a cryo-holder and cryo-transfer stage that was cooled by liquid nitrogen. Imaging was performed using a FEI Tecnai 12 TWIN Transmission Electron Microscope (100 kV) and images were recorded by a 16 bit 2K × 2K FEI Eagle bottom mount camera. To prevent sublimation of vitreous water, the cryo-holder temperature was maintained below −170 °C during the imaging process.