Naked DNA may be seen using scanning transmission EM approach with “Z-contrast.” Mercury-labeled DNA was spread on 1.2 nm thick carbon film and imaged using Titan STEM (FEI, Netherlands) from EICN/CNSI. The beaded structure represents the distribution of individual mercury atoms along the DNA. Image by RS.
Individual cells may be visualized using a classical plastic embedding approach. Image by RS.
Tight junctions keep cells together creating a tissue. Classical plastic-embedding. Image by RS.
Sample preparation is very important in EM. We use the most advanced technology to produce very thin, ultrastable carbon film used in many EM applications. This apparatus was designed and built by Sergey Ryazantsev with support from an NSF instrumentation grant (Reid Johnson PI). Electron gun shown on the pictures is capable of producing carbon films from 1.2 to 2.8 nm thick in a very controllable manner. Images, design, construction by RS.
A combination of EMi proprietary ultrathin carbon film and grids with carbon “holey film” provides the best possible condition for imaging. The carbon film attaches directly to the carbon “holey film,” which provides stability under a wide range of conditions.
In cryo-immuno localization approach, antigen is visualized using a combination of specific primary antibodies and gold-labeled secondary antibodies. Using antibodies with different gold particles, it is possible to co-localize up to 3 antigens in the same sample. Images, graphics by RS.
Negative staining is commonly used to unveil the fine structure of small macromolecules such as this dimeric aminoacylase III, total molecular weight of 70 kDa.
Example of good negative staining, GroE from Thermus thermophilus stained by uranyl-acetate. Image by RS.
High-resolution shadowing permits visualization of “difficult” samples such this DNA. Resolution of 0.8 nm allows recognition of a helical structure of DNA. Image by RS.
Single-particle 3D reconstruction permits creation of a 3D model from ordinary negatively stained samples. It is especially useful when other methods fail as shown in above case: human IgG2. Segmentation revealed most of the IgG molecule parts including V and C-parts of Fab and Fc subdivision. EMi encourages users to try this approach.
April 21, 2011