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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. In life science, negative staining with heavy metal salts such as uranyl acetate produce high contrast and protects the molecule from collapsing. Instead of the molecule (with its interior density variations) only a cast of exterior surface of the molecule is imaged, and only its shape can be reconstructed. However, the vitrification process enables the structure of macromolecules and the cellular architecture to be studied in a frozen hydrated near native state. For small objects (isolated macromolecules in a suspension organelles, small cells (a few micrometers)) we use a plunger (rapid freezing of the sample by quickly thrusting the grid into liquid ethane at liquid nitrogen temperature). In material science, structure and morphology of the nanoparticles/microparticles have grate influence on the macromolecular properties of the material, so the TEM technique is very important in investigations of solid-state materials. TEM observations on material samples reveal the nanostructure or microstructure of the material, characterized by an amorphous or crystalline nature. In crystalline materials, HRTEM bright field images show the typical lattice fringes patterns, that correspond to a group of atomic planes within the particle. Crystal structures of nanometer sized crystalline samples can be determined via structure factor amplitude information from single-crystal electron diffraction data (Selected Area Diffraction, SAD) or structure factor amplitude and phase angle information from Fourier transforms of HRTEM images of crystallites. [ For an overview of different specimens investigated, examples are available for viewing: Slideshow ] 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. Inmunoelectron 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. Three-dimensional EM reconstruction of macromolecular assemblies 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”. [ For an overview of software tools for molecular microscopy, a wikipedia article is available: 3D EM Reconstruction Software ] 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 -> SINGLE-PARTICLE RECONSTRUCTION TECHNIQUE. CIC bioGUNE Structural Biology Unit - Laboratory 3: Mikel Valle´s LAB [ For an expanded overview of CIC bioGUNE Electron Microscopy Unit, a pdf document is available for viewing: pdf ] 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) -> TOMOGRAPHIC RECONSTRUCTION TECHNIQUE. CIC bioGUNE Structural Biology Unit - Laboratory 7: Nicola G. A. Abrescia's LAB