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Histological techniques.

6. ELECTRON MICROSCOPY


When very small tissular structures, under the light microscope resolution power, are going to be visualized, such us some organelles, membranes, macromolecular complexes or viruses, we need to make use of electron microscopes. They were invented around 1950s and adapted to study biological samples shortly after. Very small cellular structures could be studied with electron microscopes, which are commonly called cell ultrastructure studies. Thus, observing cell ultrastructure means visualizing the cell with an electron microscope. The resolution power of electron microscopes may be as small as 1 nm because they use electron beams, instead of visible light. Electrons have shorter wavelength than visible light and therefore permit several million times magnification.

Electron microscopes do not have glass lenses, they use magnets instead, that work as magnetic lenses by concentrating the electron beam emitted by an electron gun. They are very large apparatuses because electrons must travel in vacuum. That is why they have large cylinders where electron beam is formed and manipulated.

Tow types of electron microscopes are commoly used in histolgy: transmission electron microscopes and scanning electron microscopes.

Transmission electron microscope

In this type of electron microscopes, a beam of electrons is produced in tungsten filament that work as a cathode. The electron beam is concentrated by electromagnets and focused on the tissue. Tissue sections have to be very thin, about tens of nanometers, to get sharp images and allow the electrons cross the whole tissue thickness. That is why they are called ultrathin sections. Previously, sections need to be treated with heavy metals like osmium, lead and uranyl, which have a similar function to dyes in light microscopy, make cellular structures visible. These metals are mainly deposit in cell membranes and macromolecular complexes. Electrons that go through the tissue are repelled by heavy metals and cannot completely cross the section. Only those electrons that go across the whole section can impact on a fluorescent screen and emit a visible light spark. The image of the section is formed with all the electrons that impact the screen. So, a black and white image is composed: white are non-repelled electrons, black are repelled electrons.

Microscopios

Main components of light microscope (on the left), transmission electron microsope (middle), and scanning electron microscopy (on the right).

Transmission electron microscopy

Transmission electron microscopy images. Increasing magnification from the left to right. Black lines of the image on the right correspond to cell membranes.

Scanning electron microscopy

Scanning electron microscopy are used to visualize sample surfaces. This is possible because electrons do not cross the sample, but interact with the surface of the sample. Samples have to be covered with a thin layer of metals over the surface. The electron beam scans the surface (that is why the name scanning microscope) and backscattered and secondary electron are emitted and impact in a detector screen, from which a digital image is formed. The complete image is gotten when the electron beam goes along the whole sample.

Samples to be observed with scanning electron microscopes are not sections, but portions of tissues. However, surfaces of sections obtained with a vibratome can be observed too.

Scanning electron microscope images

Scanning electron microscope images of the central canal of a lamprey spinal cord. Cilia can be observed and small microvilli at the apical domain of ependymal cells (see other scanning electron microscope images).


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Updated: 13-05-2019. 16:53