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In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 µm wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM's widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 Å resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.
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Contact Angle Dynamics during the Evaporation of Water from Hydrophilic and Hydrophobic Graphene Surfaces
For this experimental study on evaporation of water from graphene, two graphene samples with different thickness and microstructure were used. Figure 1 shows the representative optical and scanning electron microscope (SEM) images of the two samples. Sample 1, shown in Figure 1a-b, is a 3 to 4 atomic layer of continuous graphene sheet grown on copper substrate via chemical vapor deposition (CVD) and was subsequently transferred to a quartz substrate using a wet chemical method reported previously [5]. The graphene thickness is at 1.2 nm to 1.4 nm, as measured by Atomic Force Microscopy. Sample 2, shown in Figure 1c-d, represents an inkjet-printed reduced graphene oxide on silicon and subsequently treated with a direct pulsed laser writing (DPLW) process for surface 3D-nanostructuring. The layer thickness is between 6 µm and 7 µm.
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- Award ID(s):
- 1651451
- PAR ID:
- 10091483
- Date Published:
- Journal Name:
- http://mnf2018.me.gatech.edu/abstracts.php
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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