The core is committed to transmission electron microscopy of samples larger than 250 KDa, with the goal of image reconstruction at better than 1nm resolution. The facility’s three electron microscopes provide a progression of capability, from the Tecnai Spirit for initial screening of negative-stained samples, to the Tecnai F20 for preliminary electron cryomicroscopy, to the advanced Titan Krios, which is dedicated to automated data collection for high-resolution analysis.
The primary techniques used here are:
Electron Cryo-Microscopy (Cryo-EM)
- unstained, unfixed samples
- single particles from solutions with potential resolution < 1nm
- helical and two-dimensional crystals
- some atomic models rival X-ray crystallography and NMR
Electron Cryo-Tomography (CET)
- 3-D maps of non-regular structures, larger cellular complexes and cell sections
- uses tilt series to generate 3-D structure
- resolution 3-4 nm for irregular structures, higher for regular structures
At this time, the MEMC is not providing data analysis services. However, some guidance and suggestions are available.
Using the facilities
Trained researchers can use microscopes themselves. Alternatively, MEMC personnel can assist researchers in collecting images.
Please contact Kelly Dryden (firstname.lastname@example.org) to discuss how best to use the facility to aid your project. A project application will be required, at which time access to the scheduling system will be provided.
Clients will need to provide a 1Tb-4Tb USB storage drive for multi-day data collection on the Titan Krios. Images collected on the Spirit or F20 will accessible by download from a remote server.
Researchers can freeze samples manually in liquid ethane or using an FEI Vitrobot. Several options are also available for negative-staining.
Typical sample requirements are 3 µL at concentrations of 0.1-1.0 mg/ml.
A 2014 review in Science (10.1126/science.1256358) by Smith and Rubinstein describes how direct electron detectors can resolve helices and beta strands with resolution of less than 4Å for some samples.
University of Virginia publications using cryo-electron microscopy
A virus that infects a hyperthermophile encapsidates A-form DNA Frank DiMaio, Xiong Yu, Elena Rensen, Mart Krupovic, David Prangishvili, Edward H. Egelman, Science 22 May 2015: vol. 348 no. 6237 pp. 914-917 DOI: 10.1126/science.aaa4181
Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Lu A, Magupalli VG, Ruan J, Yin Q, Atianand MK, Vos MR, Schröder GF, Fitzgerald KA, Wu H, Egelman EH. Cell. 2014 Mar 13;156(6):1193-206. doi: 10.1016/j.cell.2014.02.008
In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery Anna B Banizs, Tao Huang, Kelly Dryden, Stuart S Berr, James R Stone, Robert K Nakamoto, Weibin Shi, Jiang He Int J Nanomedicine. 2014; 9: 4223–4230. doi: 10.2147/IJN.S64267