: An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification (>/=200,000x) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The "vitreous" nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be =2-3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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