The objective is to form an accurate three-dimensional (3D) representation (3D map) of the biological object, which ideally reveals not only its shape but also its interior density variations. Electron microscopy bridges a gap of several orders of magnitude that is left between X-ray crystallography and light microscopy. Three-dimensional information is normally obtained by interpreting micrographs as projections.
Three-dimensional imaging with the electron microscope follows two different methodologies, which are divided according to the size (physical dimensions) range of the object and its degree of “structural uniqueness”.
1.1.- Single-particle reconstruction technique:
On the one hand, we have macromolecular assemblies (in the size range of 5-50 nm), which exist in many structurally identical “copies” (identical views when placed on the support in the same orientation), (example. Ribosome).
For objects that have identical structure by functional necessity, powerful averaging techniques can be used to eliminate noise and reduce radiation damage.
1.2.- Tomographic reconstruction technique:
On the other hand, we have cell components (in the size range of 100-1000 nm), which possess a unique structure, (example. Mitochondrion). For objects that may vary from one realization to the next can only be visualized as “individuals” (by obtaining one entire projection series).
2.- Qualitative-descriptive interpretations of TEM images
By far the largest numbers of applications of electron microscopy are concerned with interpretations of the images on a qualitative-descriptive level, structure of lipid membranes (monolayers or bilayers), protein labeling, nanoparticles dispersion, crystalline or amorphous solid state, polymer conformations, etc. In those cases where quantitative measurements are obtained, they normally relate to distances, sizes, numbers of particles, etc.
3.- Inmuno-electron Microscopy technique
This technique allows the investigator to identify antibody/antigen complexes that localize to a particular subcellular organelle or compartment by using a tag (heavy metals: Colloidal gold). A single molecule of a particular antigen can be localized by a tagged antibody or series of antibodies.
The most common strategy for antigen localization (antibody labelling) is the indirect method. A tissue antigen is exposed to a primary antibody that has been made to bind the antigen. After binding of the primary antibody, a tagged secondary antibody is exposed to the bound antigen-antibody complex. The result is a two-layered antibody sequence with an attached tag.
4.- Electron Energy Loss Spectroscopy (EELS)
When an electron beam strikes a specimen, some of the kinetic energy is converted into various types of X rays, visible light, and heat. Some electrons may be transmitted through the specimen with the loss of some energy (inelastically scattered) or no loss of energy (elastically scattered). Other electrons may be given off from the top of the specimen as high energy (backscattered) electrons or lower energy (auger, secondary) electrons. These electrons with loss of energy (inelastically scattered transmitted electrons) may be separated into various energy levels in an electron energy loss spectrometer (like Omega filter) for determination of elemental composition.