Ultrascan 4k

SEM
analysis was performed
using a Quanta 450 FEG SEM commonly without prior metal deposition.
TEM was carried out on an FEI Tecnai F20 microscope, operating at
200 kV, and equipped with a Schottky field emission gun and a twin-blade
anticontaminator. All images were recorded using a Gatan 4K Ultra
Scan charge-coupled device camera. Since these organic material films
are sensitive to the electron beam irradiation, the films were normally
previewed rapidly at a dose of 20 e^– nm^–2. Selected areas were then imaged at a dose level of 200 e^– nm^–2, which did not cause radiation damage. A
drop of a solution of each compound in an organic solvent such as
CH₂Cl₂ or n-hexane was placed
on either ITO substrates for SEM or carbon-coated Cu grids for TEM.
After complete evaporation of the solvent in vacuum, the sample was
heated and cooled as described for the POM experiments.

Kinesin was desalted into EM buffer BRB80 with 0.05% (vol/vol) Nonidet P-40 (11332473001; Roche) and 2 mM DTT, supplemented with 0.5 mM ATP, using a Zeba spin desalting column and diluted to 1 mg/ml in the foregoing buffer supplemented with GTP to 1 mM (to maintain microtubule dynamics while making grids). After dilution, kinesin was spun at 80,000 × g at 4°C to remove any aggregates. Microtubules were diluted to 0.5 mg/ml in EM buffer supplemented with 1 mM GTP and applied to a glow-discharged C-flat grid (Protochips, Raleigh, NC) in the chamber of a Virtrobot (Mark II; Maastricht Instruments, Maastricht, Netherlands). Microtubules were allowed to adhere to the grid for 30 s. Kinesin was then applied to the grid twice, 4 μl each addition, briefly manually blotting after the first addition, to ensure complete decoration of the microtubules. The grid was then blotted for 4 s and plunged into ethane slush. Micrographs were collected on a Tecnai F20 transmission electron microscope (FEI, Hillsboro, OR) operating at 120 kV and equipped with a Gatan 4k Ultrascan charge-coupled device. Micrographs were collected using Leginon (Suloway et al., 2005 (link)) with a dose of 20 e⁻/Ų and nominal magnification of 80,000×, giving a final size of 1.37 Å/pixel.