Pelco easieglow

Cryogenic transmission electron microscopy samples were prepared by rapid immersion in liquid ethane using a Vitrobot Mark IV (FEI; Hillsboro, OR, USA) set to room temperature and 95% humidity. Quantifoil 200 mesh R1.2/1.3 TEM grids (Electron Microscopy Science; Hatfield, PA, USA) were rendered hydrophobic by glow-discharging for 30 seconds at 15 mA with a PELCO easieGlow (Ted Pella; Redding, CA, USA). Samples (3 μL) were spotted on grids, incubated in the Vitrobot chamber for 10 seconds, briefly blotted with Whatman 595 filter paper, and then plunged into ethane. Grids were imaged using a 200 kV Thermo Fisher Scientific Talos Arctica G3 and SerialEM software (Boulder Laboratory for 3D Electron Microscopy of Cells; Boulder, CO, USA) under low-dose conditions. To align the microscope, a cross-gradient TEM grid under parallel illumination conditions at spot size 3 with the 70 μm condenser and 100 μm objective aperture was used. A Ceta CCD camera (FEI; Hillsboro, OR, USA) at −3 μm defocus, 92,000× nominal magnification corresponding to a pixel size of 1.6 nm with a total dose of 62 e-/Ų was used to acquire images. Intermediate magnification images were acquired at 85,000× nominal magnification at −15 μm defocus.

Cryogenic TEM cryogrids were prepared by rapid immersion in liquid ethane using a Vitrobot Mark IV (FEI, Hillsboro, OR) set to room temperature and 95% humidity. Quantifoil 200 mesh R1.2/1.3 TEM grids (Electron Microscopy Science, Hatfield, PA) were rendered hydrophobic by glow-discharging for 30 seconds at 15 mA with a PELCO easieGlow (Ted Pella, Redding, CA). Before cryo-plunging, samples (3 μL) were applied to the carbon side of the TEM grid and then incubated in the Vitrobot chamber for 10 seconds. Samples were blotted for 2–4 seconds with Whatman 595 filter paper. Cryogrids were imaged with a 200 kV Thermo Fisher Scientific Talos Arctica G3 under low-dose conditions using the software platform SerialEM (Boulder Laboratory for 3D Electron Microscopy of Cells, Boulder, CO). The microscope was aligned using a cross-gradient TEM grid under parallel illumination conditions at spot size 3 with the 70 μm condenser and 100 μm objective aperture. Images were acquired with a Ceta CCD camera (FEI, Hillsboro, Oregon) at −3 μm defocus, 92,000× nominal magnification corresponding to a pixel size of 1.6 nm with a total dose of 62 e-/Ų. Contours of 200 randomly selected nanofibers were manually traced using representative cryogenic TEM images of LVFF 25 mole % nanofibers. Measurements were input to calculate nanofiber length based on methods previously described. ^{[61 (link)]}