Diseases and Management of Crops under Protected Cultivation

(Diseases and Management of Crops under Protected Cultivation)
The optimal resolving power for a light microscope is obtained with ultraviolet illumination
( = 365) if a system with the optimal NA is used (1.4).
In this example
R = 130.4 nm
In the visible region of the spectrum, blue light has the next shortest wavelength, then
green and finally red. If white light is used for illumination then the applicable wavelength is that for
green. This is in the middle range of the visible spectrum and the region of highest visible
sharpness.
Improvement of resolving power
Due to this limitation of resolving power by light microscopy, other sources of illumination,
with shorter wavelengths than visible light, have been investigated. Early experiments using X-rays
of extremely short wavelength were not pursued further because of the inability to focus these
rays. The first breakthrough in the development of the electron microscope came when Louis de
Broglie advanced his theory that the electron had a dual nature, with characteristics of a particle or
a wave. The demonstration, in 1923 by Busch, that a beam of electrons could be focused by
magnetic or electric fields opened the way for the development of the first electron microscope, in
1932, by Knoll and Ruska. Although the initial development of the electron microscope, in
Germany, was followed by technical improvements in America, the first commercially available
apparatus was marketed by Seimens.
Specimen preparation for TEM
The greatest obstacle to examining biological material with the electron microscope is the
unphysiological conditions to which specimens must be exposed.
Since the material must be exposed to a very high vacuum ( to Torr) when being
examined, it must be dried at some stage in its preparation. The biological specimen must be
stabilized (or fixed) so that its ultrastructure is as close to that in the living material when exposed
to the vacuum.
The limited penetrating power of electrons means that the specimens must be very thin or
must be sliced into thin sections (50 - 100 nm) to allow electrons to pass through.
Contrast in the TEM depends on the atomic number of the atoms in the specimen; the
higher the atomic number, the more electrons are scattered and the greater the contrast.
Biological molecules are composed of atoms of very low atomic number (carbon, hydrogen,
nitrogen, phosphorus and sulphur). Thin sections of biological material are made visible by
selective staining. This is achieved by exposure to salts of heavy metals such as uranium, lead
and osmium, which are electron opaque.
Fixatives are used to prevent autolysis, change in volume and shape and preserve various
chemical constituents of the cell.
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