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19
Notes
ELECTRON MICROSCOPY
19.1 INTRODUCTION
Electron Microscopes were developed due to the limitations of Light Microscopes
which are limited by the physics of light to a resolution of about 0.2 micrometers.
In the early 1930s this theoretical limit had been reached and there was a
scientific desire to see the fine details of the interior structures of organic cells
(nucleus, mitochondria...etc.). This required 10,000x plus magnification which
was just not possible using Light Microscopes. The Transmission Electron
Microscope (TEM) was the first type of Electron Microscope to be developed
and is patterned exactly on the Light Transmission Microscope except that a
focused beam of electrons is used instead of light to see through the specimen.
The electron microscope was invented in 1931 by Germans Ernst Ruska and
Max Knoll. Ernst Ruska later received Nobel Prize for his work in 1986.
Conventional transmission electron microscope (TEM) today can achieve a
resolution of 0.05nm.
OBJECTIVES
After reading this lesson, you will be able to:
z explain the principle of electron microscopy
z describe the tissue processing for electron microscopy.
Histology and Cytology source. The electron beam is accelerated by an anode typically at +100 keV (40
to 400 keV) with respect to the cathode, focused by electrostatic and
electromagnetic lenses, and transmitted through the specimen that is in part
transparent to electrons and in part scatters them out of the beam. When it
emerges from the specimen, the electron beam carries information about the
structure of the specimen that is magnified by the objective lens system of the
microscope. The spatial variation in this information (the image) may be
Notes viewed by projecting the magnified electron image onto a fluorescent viewing
screen coated with a phosphor or scintillator material such as zinc sulfide.
Alternatively, the image can be photographically recorded by exposing a
photographic film or plate directly to the electron beam.
Histology and Cytology fixation used is double fixation. It involves primary fixation in an aldehyde
followed by secondary (post) fixation in osmium tetroxide. Glutaraldehyde is
the most popular aldehyde for fixation of tissues for electron microscopy as it
reacts rapidly with proteins and stabilises structures by cross-linking before there
is any opportunity for extraction by the buffer . Hence more ground substance
of the cytoplasm (glycogen) and of the extracellular matrices is preserved. But,
glutaraldehyde alone is not an adequate fixative, since certain cell components
Notes especially lipids, are not fixed and may be extracted during dehydration,
therefore secondary fixation is required using osmium tetroxide. Depth of
penetration of glutaraldehyde is 2 - 3 mm / hour and of osmium tetraoxide is
1mm/hour.
Dehydration
The aim of dehydration is to remove all the free water in the fixed tissue and
replace it with a solution miscible with embedding medium. We usually use
organic solvents like methanol, ethanol or acetone. It starts with distilled water
to 40% ethanol and then through a series of increasing concentrations of ethanol
to 100% ethanol.
Protocol for dehydration in ethanol
1. 40% ethanol, 5 min.
2. 70% ethanol, 10 min.
3. 90% ethanol, 10 min
4. 100% ethanol, 3 x 10 min.
5. If ethanol is miscible with the embedding medium then tissue is directly
transferred to it. Otherwise, another transitional fluid may be required.
Epoxypropane is most commonly used transitional fluid.
Embedding Media
The embedding media for EM are resins, polyester resins and methacrylates. For
general electron microscopy epoxy resins have most properties required.
Epoxy resin
Characteristics
Embedding
The embedding is carried out in polythene capsules or flat embedding moulds.
The capsules are filled with warm bubble-free raisin using a plastic pipette. No
air bubbles should be introduced in the medium. Labeling is done at this point.
Notes
It is best to write the identifier on a piece of paper and embed it along with tissue
using marking ink or pencil.
Sectioning
Getting an ultra-thin section is the pre-requisite for EM. It should be approximately
60 nm in thickness. Routine microtomes cannot produce such thin sections, thus
special microtome known as ultramicrotome is used which needs special
training. Glass knives or diamond knives are used to cut ultra-thin sections on
these ultra microtomes.
TERMINAL QUESTIONS
1. What is the principle of EM?
2. What are the types of electron microscope?
3. What is the procedure for processing tissue for EM?
4. What are the applications of EM?
19.1
1. Electron beam
2. Electromagnetic coils
3. Absorption & Scattering
4. Scanning electron microscopy
5. Double fixation
6. Gluteraldehyde
7. Epoxypropane
8. 60nm