With the help of an electron microscope, it becomes possible to carry out a detailed study of the toxic lesions in order to understand the toxicological events.
- Electron Tomography and Sub-cellular Structure
Electron tomography is a technique which is used to visualize and investigate the three-dimensional structural information about the sub-cellular macromolecular specimens. It is a three-dimensional electron microscopy technique used for sub-cellular imaging.
Principles of Function
- Electrostatic Interactions
In an electron microscope, electrostatic and electromagnetic lenses are used to selectively control and focus the electron beam to generate images. In atoms, with the increase in atomic numbers the Coulomb forces increase proportionally. As a result, the areas with heavy localized atoms appear darker in comparison to the light atoms. Therefore, on interacting with an atom, the probability of deviation of an electron from its direct path depends upon the number of charges carried by the atom.
When an electron drops to a lower energy shell to fill the holes, the difference in the energies between the two shells gets emitted in the form of high-energy electromagnetic radiation. This electromagnetic radiation is emitted in the form of an x-ray quantum with a characteristic energy. Every element has a distinct number and arrangement of electrons in its shells. Therefore, the energy differences between their shells and the resulting x-ray emissions also vary and are the characteristic properties of the element. When the x-ray spectrum is recorded valuable information about the element can be obtained.
- Specimen Preparation
The specimen to be observed by an electron microscope needs to be processed prior to its use. The specimen preparation technique depends upon the specimen, the analysis requirements and the microscope type. The preparation techniques include Cryofixation, Dehydration, Embedding, Sectioning, Straining and the freeze-fracture methods.
- Transmission Electron Microscopy
Transmission Electron Microscope (TEM), the earliest form of electron microscope involves the transmission of a high voltage electron beam formed by the magnetic lenses passing through a very thin layer of the specimen. This electron beam contains information about the structure of the specimen. There is a series of magnetic plates that magnify the spatial variations in the image. It then hits a fluorescent screen, photographic plate or a light-sensitive sensor where it gets recorded. This image can now be displayed on the screen. The TEM technique facilitates the visualization of minute and precise details about the structure of the specimen.
- Scanning Electron Microscopy
In Scanning Electron Microscopy (SCM), three-dimensional topographical information about the specimen is gathered by tracing and detecting the secondary electrons that are emitted out from the surface of the specimen when it gets excited due to the primary electron beam. The accelerating voltage of the electron beam is kept much lower as compared to TEM since any penetration of the beam into the sample is undesirable. While the resolution of images generated from TEM is slightly better than SEM, for bulk samples SEM provides images with much greater depth of view.
- Reflection Electron Microscopy
The Reflection Electron Microscopy (REM) is used to determine the topography, crystal structure and the chemical composition at the surface layer of the specimen. It employs the techniques of diffraction, spectroscopy and imagining.
- Scanning Transmission Electron Microscopy
In this technique, the specimen is bombarded with a finely focused electron beam. This bombardment can take place either all across the specimen or its optical axis. The secondary transmitted electrons that get back scattered and diffracted as well as its characteristic x-ray spectrum is recorded. With the help of the X-ray spectrum obtained, the chemical composition of the micrometer and sub-micrometer domains of the specimen can be obtained. Apart from this the electron diffraction pattern helps to achieve its structural characterization.