Electron Microscopic Examination of Microorganisms
Electron microscopic examination of microorganisms
Depending upon the microscope used and the preparation technique, an entire intact organism, or thin slices through the interior of the sample can be examined by electron microscopy. The electron beam can pass through very thin sections of a sample (transmission electron microscopy) or bounced off of the surface of an intact sample (scanning electron microscopy). Samples must be prepared prior to insertion into the microscope because the microscope operates in a vacuum. Biological material is comprised mainly of water and so would not be preserved, making meaningful interpretation of the resulting images impossible. For transmission electron microscopy, where very thin samples are required, the sample must also be embedded in a resin that can be sliced.
For scanning electron microscopy, a sample is coated with a metal (typically, gold) from which the incoming electrons will bounce. The deflected electrons are detected and converted to a visual image. This simple-sounding procedure requires much experience to execute properly.
Samples for transmission electron microscopy are processed differently. The sample can be treated, or fixed, with one or more chemicals to maintain the structure of the specimen. Chemicals such as glutaraldehyde or formaldehyde act to cross-link the various constituents. Osmium tetroxide and uranyl acetate can be added to increase the contrast under the electron beam. Depending on the embedding resin to be used, the water might then need to be removed from the chemically fixed specimen. In this case, the water is gradually replaced with ethanol or acetone and then the dehydrating fluid is gradually replaced with the resin, which has a consistency much like that of honey. The resin is then hardened, producing a block containing the sample. Other resins, such as Lowicryl, mix easily with water. In this case, the hydrated sample is exposed to gradually increasing concentrations of the resins, to replace the water with resin. The resin is then hardened.
Sections a few millionths of a meter in thickness are often examined by electron microscopy. The sections are sliced off from a prepared specimen in a device called a micro-tome, where the sample is passed by the sharp edge of a glass or diamond knife and the slice is floated off onto the surface of a volume of water positioned behind the knife-edge. The slice is gathered onto a special supporting grid. Often the section is exposed to solutions of uranyl acetate and lead citrate to further increase contrast. Then, the grid can be inserted into the microscope for examination.
Samples can also be rapidly frozen instead of being chemically fixed. This cryopreservation is so rapid that the internal water does not form structurally disruptive crystals. Frozen thin sections are then obtained using a special knife in a procedure called cryosectioning. These are inserted into the microscope using a special holder that maintains the very cold temperature.
Thin sections (both chemically fixed and frozen) and whole samples can also be exposed to antibodies in order to reveal the location of the target antigen within the thin section. This technique is known as immunoelectron microscopy. Care is required during the fixation and other preparation steps to ensure that the antigenic sites are not changed so that antibody is still capable of binding to the antigen.
Frozen samples can also be cracked open by allowing the sample to strike the sharp edge of a frozen block. The crack, along the path of least chemical resistance, can reveal internal details of the specimen. This technique is called freeze-fracture. Frozen water can be removed from the fracture (freeze-etching) to allow the structural details of the specimen to appear more prominent.
Samples such as viruses are often examined in the transmission electron microscope using a technique called negative staining. Here, sample is collected on the surface of a thin plastic support film. Then, a solution of stain is flowed over the surface. When the excess stain is carefully removed, stain will pool in the surface irregularities. Once in the microscope, electrons will not pass through the puddles of stain, producing a darker appearing region in the processed image of the specimen. Negative staining is also useful to reveal surface details of bacteria and appendages such as pili, flagella and spinae. A specialized form of the staining technique can also be used to visualize genetic material.
Electron microscopes exist that allow specimens to be examined in their natural, water-containing, state. Examination of living specimens has also been achieved. The so-called high-vacuum environmental microscope is finding an increasing application in the examination of microbiological samples such as biofilms .
See also Bacterial ultrastructure; Microscope and microscopy