Electron Microscopy and Autoradiography

Objectives: Upon completion of this lecture, the student should be able to:
1- Explain the main components of SEM & TEM.
2- Discus the differences between light and electron microscopy.
3- Differentiate between SEM and TEM images.
4- Describe briefly how to use the Autoradiography method for macromolecules localization.
Transmission and scanning electron microscopes are based on the interaction of electrons and tissue components. The wavelength in the electron beam is much shorter than that of light, allowing a thousand-fold increase in resolution.
Transmission Electron Microscopy
The transmission electron microscope (TEM) is an imaging system that permits very high resolution (3 nm) (Figure 1a). This high resolution allows magnifications of up to 400,000 times to be viewed with details. Unfortunately, this level of magnification applies only to isolated molecules or particles. Very thin tissue sections can be observed with details at magnifications of up to about 120,000 times.
The TEM functions on the principle that a beam of electrons can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses. The beam is produced by a cathode at the top of the instrument and passes down through the chamber in a vacuum. Because electrons change their path when submitted to electromagnetic fields, the beam can be focused by passing through electric coils which can be considered as electromagnetic lenses.
The first lens is a condenser focusing the beam of electrons on the specimen section. Some electrons interact with atoms in the section and their course is modified, while others simply cross the specimen without interacting. Electrons passing through the specimen reach the objective lens, which forms a focused, magnified image that is then magnified further through other lenses and captured on a viewing screen. The image of the specimen shows areas of white, black, and shades of gray corresponding to areas through which electrons readily passed (appearing brighter or electron lucent) and areas where electrons were absorbed or deflected (appearing darker or more electron dense). To improve contrast and resolution in TEM, compounds with heavy metal ions (like osmium tetroxide, lead citrate) are often added to the fixative or dehydrating solutions used to prepare the tissue. This will bind cellular macromolecules, increasing their electron density and visibility.