The Electron Microscopy Core Facility provides critical ultrastructural skills, including immunocytochemistry, in addition to conventional EM techniques. The methodology can be tailored to specific needs. The Facility includes a T12 microscope, a fully equipped sample processing laboratory, a sectioning room, and a dark room. The Facility offers complete transmission electron microscopic services including sample processing, ultramicrotomy, film developing, and photographic printing, as well as immunocytochemistry. Intramural collaboration is available. We welcome trained EM users as well as researchers who wish to be trained. Scroll to read more about the methodologies involved.
CCMSB, Room 111G
In transmission electron microscopy (TEM), a high-energy electron beam is used to examine the structures of molecules, down to the level of atomic details. The electron beam passing through a very thin specimen projects a two-dimensional image of the sample onto the detector (a charge-coupled device; CCD or a direct electron detector; DED). Computational algorithms are used to align the two-dimensional image projections to generate a three-dimensional reconstruction which in turn is used for atomic model building.
Cryogenic Electron Microscopy (Cryo-EM) is an approach that allows the observation of hydrated biological specimens in their native environment at cryogenic temperatures in TEM. Cryo-EM broadly encompasses three different approaches: electron crystallography, single-particle cryo-EM, and electron cryotomography. Recent technological advancements in cryo-EM have ushered in a new era of structural biology enabling exclusive views of the complex biological molecules. Atomic details of biomolecules allows us to better understand physiological phenomena that govern life and aid in drug design and development.
CWRU cryo-EM facility houses state-of-the-art electron microscopes in a newly renovated space (Titan Krios G3i+GIF+K3 camera, FEI Tecnai F20+Tvips F416&DE20 camera, FEI Tecnai T12+Gatan895 camera). The facility is set up to be used with flash-frozen (single particle, cryo-ET and crystallography) samples. The Facility also offers complete conventional TEM services including sample processing, ultramicrotomy, film developing, and photographic printing, as well as immunocytochemistry. Intramural collaboration is available. We welcome trained EM users as well as researchers who wish to be trained.
Single-particle cryo-EM can provide structural information for a large variety of biological molecules without the need to produce crystals. Proteins from 40 kDa to several MDa in size can be studied by this methodology. Very little sample is required for this technique. For cryo-EM the specimen, typically a purified protein sample, is embedded in vitreous ice on a cryo-EM grid, and is kept at cryogenic temperatures while images are recorded by the electron microscope. After completing the imaging, single-particle reconstruction methods are used to solve the structure of the protein. Current resolution for single particle cryo-EM is in the 1.65~3.5 Å range.
Cryo-electron tomography (Cryo-ET) can unveil the detailed 3D structures of cellular and subcellular macromolecular objects. High pressure-freezing technique for cells and tissue can provide exceptional preservation of 100µm large samples in the native state. Single cells can be vitrified by plunging them into liquid ethane. This allows studying macromolecular complexes in their cellular environment, which provides a deep insight into cellular processes at the molecular level. 2D images (projections) of the sample collected at different angles allow the 3D structure to be reconstructed by the back-projection method. Resolution for cryo-electron tomography is in the 4-30 Å range.
Electron Crystallography is coming into the scope of the structural biologists and chemists by its capability in determining the structures of small crystals, named MicroED technology which developed by Tamir Gonen’s research group in 2013. The method involves shooting electrons at tiny protein crystals under cryogenic conditions and collecting the resulting electron diffraction patterns. Electrons interact with matter more strongly than x-rays, which is why it’s possible to obtain useful data from very tiny crystals. Resolution for microED is in the 0.6-2.0 Å range.