Electron microscopy

The electron microscopy is a type of microscopy that uses a beam of electrons instead of light to create an image of the specimen. Electromagnets, instead of glass lenses, control focus, illumination and magnification. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail. Objects smaller than 0.2 m m such as viruses, or the internal structures of cells can be examined. They are large, expensive pieces of equipment, generally standing alone in a small, specially designed room and requiring trained personnel to operate them.

Transmission Electron Microscope (TEM)

Transmission electron microscopy (TEM) involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the structure of the specimen (Fig. 8). The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer. Transmission electron microscopes produce two-dimensional, black and white images. Resolution of the TEM is also limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome or limit these aberrations. Software correction of spherical aberration has allowed the production of images with sufficient resolution.

Fig. 8. Transmission electron microscope

Specimens are prepared by negative staining, shadowing with metal or free-etching and can resolve objects as close as 2.5 nm.

Preparation of specimens for TEM:

•  Specimens must be around 20-100 nm thick and should be able to maintain its structure when bombarded with electrons under high vacuum.

•  Thin slice can be cut with the necessary support like plastic

•  After fixate with chemicals like glutaraldehyde or osmium tetroxide to stabilize cell structures, the specimen is dehydrated with organic solvents (e.g., acetone or ethanol)

•  Specimen is soaked in un-polymerized, liquid epoxy plastic until it is completely permeated, and then the plastic is hardened to form a solid block. Thin sections are cut from this block with a glass or diamond knife using a special instrument called an ultra microtome

•  Specimens are soaked with solutions of heavy metal salts like lead citrate and Uranyl acetate (make them more electron opaque), thus increasing the contrast in the material

•  Stained thin sections are then mounted on tiny copper grids and viewed.

Three types:

1.  Negative staining:

• •  Specimen is spread out in a thin film with either phosphotungstic acid or uranyl acetate

•  Heavy metals do not penetrate the specimen but render the background dark, whereas the specimen appearslight in photographs

•  Excellent way to study the structure of viruses, bacterial gas vacuoles and other similar objects

2.  Shadowing:

• •  It is coated with a thin film of platinum or other heavy metals by evaporation at an angle of about 45⁰ from horizontal so that the metal strikes the microorganism on only one side

•  The area coated with metal scatters electrons and appears light in photographs. Whereas the uncoated side and the shadow region created by the object is dark

•  Particularly useful in studying virus morphology, bacterial flagella and plasmids

3.  Freeze-etching technique:

• •  Disclose the shape of organelles within microorganism by this technique

•  Cells are rapidly frozen in liquid nitrogen and then warmed to -100°C in a vacuum chamber

•  A knife that has been pre cooled with liquid nitrogen (-196°C) fractures the frozen cells, which are very brittle and break along lines by greatest weakness

•  Specimen is left in the high vacuum for a minute or more so that some ice can sublimate away and uncover more structural detail

•  Finally, the exposed surfaces are shadowed and coated with layers of platinum and carbon to form a replica of the surface

•  After the specimen has been removed chemically, this replica is studied I the TEM and provides a detailed, three-dimensional view of intracellular structure.