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Immunoelectron microscopyThe first prototype of the electron microscope was put together in 1931 by the physicist Ernst Ruska and the electrical engineer Max Knoll, and for this work Ernst Ruska was awarded the Nobel Prize in 1986 (The Nobel Prize in Physics 1986). Ernst Ruska continued his work and improved the magnification; during the years 1937 and 1938 the first prototype was produced that became the first serially produced electron microscope. TechnologyElectron microscopy uses a focused beam of accelerated electrons instead of light in order to obtain an image of a specimen. There are two variants of the electron microscope: the scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The images are obtained by detecting signals resulting from the interaction of electrons with the surface of a specimen when using SEM, and from the electrons passing through an ultra-thin specimen when using TEM. The wavelength of accelerated electrons is shorter than that of visible light, which allows for better spatial resolution. For comparison, the resolving power (i.e. the shortest distance between two points in the specimen that can be visualized separately), of the human eye is 0.2 mm of a light microscope is 0.2 µm and that of electron microscopes is 0.2-2 nm. The contrast of the image in the electron microscope comes from the strength of interaction between the accelerated electrons and the atoms in the specimen. For biological specimens, usually composed of lightweight elements, the contrast in TEM is increased by adding heavy elements (osmium, lead, or uranium), which conjugate preferentially with distinct chemical molecules (i.e. lipids) thus allowing visualization of various organelles (i.e. membranes). Sample preparationThe need for ultrathin specimen in TEM requires special protocols producing very hard blocks that allow cutting of 50-70 nm thick sections. This can be obtained by embedding the sample in special plastic resins that harden after polymerization, or by rapidly freezing down the specimen in liquid nitrogen and cutting it at ultracold temperature (cryo-ultramicrotomy). Perfect preservation of the live cells ultrastructure and of epitope antigenicity put strict demands on the fixation and embedding methods, on the chemicals, and on the specificity of the antibodies. For example, to insure optimal cell morphology, rapid cooling of the specimen in the presence of cryoprotectants or fixation in a combination of glutaraldehyde with formaldehyde buffers is employed. Three general methods are used, depending on the amount and the intracellular location of the antigen and on the fixation technique: (i) cryo-ultramicrotomy, (ii) pre-embedding, and (iii) post-embedding. All three methods include fixation, sectioning, blocking, labeling, and contrasting in different order before TEM examination. An overview of the methods is given in Figure 1.
Figure 1. Flow chart for immunoelectron microscopy methods. DetectionImmunoelectron microscopy combines the ability of an antibody to specifically bind a protein with the high spatial resolution of an electron microscope in order to determine the protein’s location at subcellular level. The visualization is performed by TEM, which gives a simple two-dimensional projection of the ultrathin sample down the optic axis. Detection of the antibody’s localization in the sample is possible by conjugating it with colloidal gold particles of known diameter, which are thus distinctly visible on the transmitted image of the sample. By using the tomography system available on modern electron microscopes, one can go beyond the two dimensional projection and get accurate 3D information on the location of the protein and with even higher resolution power. Specific examplesMost commonly immunoelectron microscopy is used to answer the question of the subcellular localization of a given protein, as in Figure 2. Under proper experimental conditions it has the potential to greatly advance the understanding of structure-function relationships. When using the post-embedding method, double labeling immunoelectron microscopy is possible by using colloidal gold of different particle sizes for the two antibodies. This permits the simultaneous localization of two antigens in the cell, albeit rigorous controls of specificity are essential. For highly specific antibodies it is also possible to semi-quantitatively analyze the resulted gold particles distribution pattern and density (morphometric analyses). Together, all these methods allow the analysis of dynamic cellular processes such as metabolic activity, differentiation, proliferation, cellular transport, etc. Immunoelectron microscopy is one of the essential tools that can fill the information gap between molecular biology and cell biology, by placing macromolecules such as proteins within a relevant cellular context.
Figure 2. Detection of IAPP amyloid fibrils in endocrine cells (pancreas). Arrows indicate gold particles (10 nm in diameter) conjugated with antibodies binding to IAPP amyloid. The dark round vesicles represent insulin granulae. Courtesy of Prof. G. Westermark, Uppsala University, Sweden. References and LinksThe Nobel Prize in Physics 1986. (n.d.). Retrieved March 24, 2014: http://www.nobelprize.org/nobel_prizes/physics/laureates/1986/ De Paul et al. (2012) Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes. In: Dehghani (eds) Applications of immunocytochemistry, InTech. Hacker C et al., Analysis of specificity in immunoelectron microscopy. Methods Mol Biol. (2014) Antibodypedia - An open-access database of publicly available antibodies and their usefulness in various applications: http://www.antibodypedia.com |
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