Electron+dense+staining+for+electron+microscopy

Electron Dense Stains
Electron dense stains are stains used in transmission electron microscopy (TEM) for the purpose of visualizing biological materials. They are salts of heavy metals such as osmium, uranium, lead, tungsten, etc. Some examples of these stains are lead citrate, sodium phosphotungstate, phosphotungstic acid, and osmium tetroxide (which functions as both a fixative and an electron dense stain).

Purpose
Biological specimens require complex, careful, and specific preparations for viewing with electron microscopy. The purpose of electron dense stains is to make biological tissue visible for TEM while keeping its structures as close as possible to that of its living state. The contrast of an image generated by TEM depends on the atomic number of the elements in the sample that the electron beam is fired at. Elements of higher atomic numbers cause more scattering of the electrons in the beam and therefore more contrast in the image. Since elements of low atomic numbers like C, O, N, or H usually comprise biological material, a stain is require in order to provide the contrast necessary to create a clear image of the sample [1]. The electron dense stains allow the viewer to see different cell constituents with different degrees of contrast depending on the specific stain and the amount of it used [1]. This allows for cell components to be located their structures to be viewed. Specific stains are chosen based on which cell parts they will associate with because they will cause those cell parts to be dense, which reflects and scatters the electrons from the beam. The rest of the electrons will pass through the specimen to create the image. The areas of higher electron density block electrons and therefore appear as a darker areas in the image because of lower electron flux [1]. The image appears on a phosphorescent screen or is recorded by a photographic plate or a high-resolution digital camera depending on which method the researcher decides to use [1]. For accurate viewing, biological materials usually need to be fixed, cut into extremely thin sections, and then stained. In some instances the last staining step is not needed. For example, if you fix your sample with osmium textroxide which is both a fixative and electron dense stain that binds to lipid bilayers, you would not need an additional stain to visualize a membrane [1].

History
The use and protocol of electron dense staining evolved over time with advances in transmission electron microscopy. The earliest electron microscopes weren't useful in viewing non-metallic specimens because the electron beam had a high current density and was very hot when concentrated into a small area. Any biological specimen would be charred by the beam and therefore unable to be studied [2]. Around 1940-1941, Hans Mahl of Germany was working with transmission electron microscopy and discovered a method of viewing the surface of an object. He found that a thin layer of lacquer could be painted on an object, dried, removed, and viewed using TEM to see a thin-film replica of the object's surface. This discovery led to the realization that objects could be coated in a thin layer of metal to increase contrast and become more observable [5]. Transmission electron microscopy finally came into use for viewing biological specimens when it was discovered that cutting the specimens into very thin slices and treating them with osmium allowed them to be observed. Since then, a number of electron dense stains and staining techniques have been developed.

Use in Recent Research
In an article entitled, "Transmission electron microscopy observation of antibody", researchers set out to achieve nanoscale observation of IgG molecules using TEM. Due to the light elemental composition of biological materials, their images have very low contrast. The researchers remedied this by using the negative, electron dense stain, sodium phosphotungstate. They were able to observe individual a front view of Y-shaped antibodies standing on mica in a vacuum and obtained a high resolution image of a side view of the antibodies [3].

Another example of TEM utilized in recent research can be found in the article, "Rapid diagnosis of plant virus diseases by transmission electron microscopy". TEM observation with negative staining can quickly and reliably detect and identify viruses/viral diseases in plants but sometimes the ultrastructural changes that have been induced need to be examined in order to make a diagnosis. Unfortunately, the preparation for observation of those changes is very time consuming and it can take several days to get results. The authors of this article compared that lengthy conventional sample prep method with a newer and quicker method utilizing microwave irradiation and negative staining. Both methods utilized electron dense stains; the conventional method used lead citrate and the microwave irradiation method used phosphotungstic acid. The microwave irradiation method was found to reduce sample prep time from 3 days to 136 minutes without causing any negative effects on the quality of the samples or their ultrastructural details. //Nicotiana tabacum// plants infected with //Tobacco mosaic virus// and //Curcurbita pe////po// plants infected with //Zucchini yellow mosaic virus// were the samples used in this investigation [6].

A third example of TEM in research can be found in "Identification of neuromuscular junctions by correlative confocal and transmission electron microscopy". Detailing the structures of neuromuscular junctions (NMJs) in muscular biopsies is a very helpful way to do research and make diagnoses, however NMJs are difficult to locate using electron microscopy alone. The researchers here developed a "correlative confocal transmission electron microscopy method" where NMJs in fixed samples are found using fluorescent stains in confocal microscopy and then the samples containing NMJs are observed using TEM. The samples here obtained from the legs of euthanized female rats were stained using the electron dense stain, osmium tetroxide. This stain was used because it provided contrast. This meant that the membranes of the samples did not need to be coated in metal and the researchers could observe an unobstructed view of pre and post-synaptic structures [4].