Transmission Electron Microscopy and Computer-Aided Image Processing for 3D Structural Analysis of Macromolecules

Abstract

The Transmission Electron Microscope (TEM) is an electron-based imaging system used to reveal the atomic or molecular details of a specimen. High-energy electrons are used to probe the object in question, resulting in the generation of a two-dimensional (2D) image of the object's three-dimensional (3D) information. The electrons in use, with typical energies of greater than 100 kV, have wavelengths less than a tenth of an Ångström, theoretically allowing for imaging resolution far below the sub-Ångström range. However, due to the presence of imperfect imaging conditions such as lens aberrations and sample irradiation, the information transferred from biological specimens via TEM has yet to reach the sub-Ångström limit. The electrons used for TEM imaging are generated at the top of the EM column by a variety of methods. Early electron microscopes utilized a tungsten filament as an affordable electron source. By heating the filament to high temperatures (2700 K), electrons of the desired energy can be generated and sent down the column. The development of lanthanum hexaboride (LaB 6) crystals was an improvement over the use of tungsten, as its lower work function of 2.5 eV (vs. 4.5 eV), and its magnitude increase in brightness (current density per unit emission angle) allowed for much better beam generation. Unfortunately, thermionic filament sources such as tungsten and LaB 6 share the common limitation of having high electron energy spreads (1.5-3.0