Specimen preparation techniques

There are many ways of preparing specimens for electron microscopy. In particular, a specimen preparation method for a biological sample perform the task of stabilizing the initially hydrated molecules, so that this sample can be placed and observed in the harsh vacuum of an electron microscope. Biological specimens at our laboratory are typically prepared using negative staining with heavy metal salts such as uranyl acetate producing high contrast, or by cryo-fixation techniques where the samples are embedded in vitreous ice in a frozen hydrated near native state.

Three-dimensional cryo-EM reconstruction of macromolecular assemblies

Cryo-transmission electron microscopy (cryo-TEM) is a low-dose imaging technique to visualize macromolecular structures, such as virus, proteins and pieces of cell organelles, in a near native state with sub-nm resolution. Depending on the specific question and or sample, two different cryo-EM data processing methodologies can be used to form an accurate three-dimensional (3D) representation (3D-map) of the biological object with sub-nm resolution, from a set of 2D noisy cryo-EM images. One of these methods is Single Particle Analysis and another method is Cryo-Electrom Tomography (cryo-ET). On the one hand, for macromolecular assemblies (in the size range of ~5-50 nm) that have identical structure by functional necessity, powerful averaging techniques can be used to eliminate noise and obtain near atomic resolution 3D maps (example. Virus, ribosomes …). On the other hand, for cell components (in the size range of ~100-1000 nm), which possess a unique structure, (example. Mitochondrion, bacteria …), they are visualized as “individuals” by obtaining one entire projection series that is aligned and reconstructed to produce a tomogram.