Electron Microscope: Principle,

Components, Specimen Preparation

and Uses
Let us make an in-depth study of the electron microscope. After reading
this article you will learn about: 1. Principle of Electron
Microscope 2. Transmission Electron Microscope (Tem) 3. Components
of Electron Microscope 4. Preparation of Specimen 5. Image Viewing,
Development and Recording Techniques 6. Use of Electron
Microscope 7. High Voltage Modern Electron Microscope 8. Scanning
Electron Microscope and 9. Phase Contrast Microscope.
Introduction to Electron Microscope:
In electron microscope, high-speed electron beam is used instead of light waves,
which are used in optical microscope. Like light, the stream of electrons has a
corpuscular and vibratory character. Electron microscope gives very high
magnification and incredibly high resolution.

The first transmission electron microscope was developed by Ernst Ruska and Max
Knoll of Germany in 1931. EM is a remarkable research tool of twentieth century. It
opened up subcellular structures, which were unknown to biologists. It can magnify
an object upto 1000000X (one million times). The photomicrographs can be further
enlarged and studied by using modern photographic techniques and computer aided
techniques.

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The electrons can be focused by electro-magnetic lenses much like the light rays.
Electron beam can vibrate like light rays but has very short wave length as compared
to light rays. Wave length of electron beam λ = 0.005 nm as compared to 550 nm of
visible light. Resolution increases with the decrease of wave length.

Resolution is dependent upon the wavelength of radiations. Smaller the radiation,

greater will be the resolution. It is inversely proportional relation. The resolution
determines the level of details that can be viewed from the specimen. It provides
remarkable pictures with fine details.

Light microscope can achieve a maximum resolution of about 0.2 µm or 200 nm.
Whereas EM can achieve a resolution of 0.10 nm which is 2000 times better than
the best resolution by light microscope. Resolution is the ability of a lens to separate
or distinguish between closely positioned small objects.
Principle of Electron Microscope:
Electrons are subatomic particles, which orbit around the atomic nucleus. When
atoms of a metal are excited by heat energy, electrons fly off from the atom. In
electron microscope, tungston is heated by applying a high voltage current, electrons
form a continuous stream, which is used like a light beam.

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The lenses used in EM are magnetic coils capable of focusing the electron beam on
the specimen and illuminating it. The strength of the magnetic lens depends upon
the current that flows through it. Greater the flow of the current, greater will be
strength of the magnetic field. The electron beam cannot pass through the glass lens.

Transmission Electron Microscope (Tem):

It consist of a system of electromagnetic lenses mounted in a column.
Components of Electron Microscope:
EM is in the form of a tall column which is vertically mounted.

It consists of the following main components:

1. Electron gun

2. Electromagnetic lenses—three sets.

3. Image viewing and recording system.

Electron gun is a heated tungsten filament, which generates electrons. Condenser

lens focuses the electron beam on the specimen. A second condenser lens forms the
electrons into a thin tight beam.

To move electrons down the column, an accelerating voltage is applied between

tungsten filament and anode. Now most EMs use accelerating voltages between 100
kV-1000 kV. Electrons also function as a source of illumination for the specimen.
High velocity electrons pass into the system of condenser lenses, which focus them
on the specimen.
The specimen to be examined must be extremely thin, at least 200 times thinner
than those used in optical microscope. Ultra thin sections of 20-100 nm are cut. The
specimen holder is an extremely thin film of carbon or collodion held by a metal
grid.

The electronic beam passes through the specimen and electrons are scattered
depending upon the thickness or refractive index of different parts of the specimen.
The denser regions in the specimen scatter more electrons and therefore appear
darker in the image since fewer electrons strike that area of the screen. In contrast,
transparent regions are brighter.

The electron beam coming out of the specimen passes down the second of magnetic
coils called objective lens, which has high power and forms the intermediate
magnified image. Finally, a third set of magnetic lenses called projector (ocular)
lenses produce the final further magnified image.
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Each of these lenses acts as image magnifier all the while maintaining an incredible
level of details and resolution. He whole image remains in focus. This image is
projected on a fluorescent screen. Below the fluorescent screen is a camera for
recording the image. These lenses provide immense magnification and resolution.

As the EM works in vacuum, the specimen should be completely dry. Air molecules
present in the column of EM scatter the electrons causing flicker in the electron
beam. Vacuum is created in two steps. Firstly, a mechanical vacuum pump is used to
create vacuum. Secondly, a diffusion pump uses a fast downward moving liquid,
either oil or mercury which traps air and gas in the column. In this way, ultra high
vacuum is created.

Preparation of Specimen:
The specimen have to be specially prepared for EM studies. There are various
techniques for studying the specimen under EM. Some of which are discussed here.

Fixation and Dehydration:

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The specimens are fixed in glutaraldehyde, osmium tetroxide to stabilize the cell
structure. After fixation, dehydration is carried out slowly with organic solvents like
acetone and ethanol.

Embedding:
Resins such as araldite and epoxy are used for this purpose. Microbes are embedded
in plastic resin. The specimen is soaked in un-polymerized, liquid epoxy plastic until
it is completely permeated and then is hardened to form a solid block.

Ultra sectioning:
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To obtain extremely thin sections from this plastic block, Ultra-microtomes with
diamond knife or glass knives are used.

Staining:
Specimens are stained with heavy metals such as lead, uranium, phosphotungstic
acid. The thin sections soaked in solutions of heavy metals like lead citrate, uranyl
acetate or osmium tetroxide is also used for staining.

There are some additional techniques for preparation and study of various
specimens and materials.

Image Viewing, Development and Recording Techniques:

The image formed in EM is real as compared to the virtual image in optical
microscope. The highly magnified image is formed below the projector lens on a
fluorescent screen. Below this screen, a camera or a film or light sensitive sensor
such as charge-coupled device (CCD) camera are placed. The image can be displayed
on computer or monitor. For direct viewing monocular or binocular viewing, lenses
are used.

The final image formed will always be in focus and needs no adjustments. The image
recording and studying have undergone revolutionary changes. Digital cameras and
computers have come to play a major role.

Instead of one picture of one section, series of sections are studied and analysed. By
computer aided averaging techniques of numerous images three- dimensional
reconstructions of cell organelles of highest clarity are developed. Tilting of
specimen also provides three-dimensional picture.

Use of Electron Microscope:

Invention of EM has come as a boon for biological sciences and industry. There is
hardly any area of science that has not gained from the use of electron microscope.
Immense magnification, high resolution has opened new vistas in research in
cellular and molecular biology.

Study of microorganisms like bacteria, virus and other pathogens have made the
treatment of diseases very effective. Fields of medicine, pathology, human anatomy
have gained immensely from electron microscope studies. Health field has benefited
tremendously. Nanotechnology studies are the result of electron microscope studies.

Science of microbiology owes its development to electron microscope. It also helps in

tumour identification, biopsy, study of cells, variety of molecules. In industry high
resolution. 2D and 3D imaging. In foresenic, mining, chemical and petrochemical
industries.
High Voltage Modern Electron Microscope:
The recently developed EMs use accelerating voltages between 500-3000 kV. These
EMs are used in study of metallurgy, biological materials and living cells. Thick
sections upto 5µ m can also be studied.
Scanning Electron Microscope:
Transmission electron microscope (TEM) and scanning electron microscope (SEM)
work on the same basic principle. TEM forms image when radiations pass and are
transmitted through the specimen. Whereas SEM produces images by detecting
secondary electrons which are emitted from the surface of the specimen due to
excitation by the primary electron beam. Therefore SEM is used to examine the
surfaces of the microorganisms in great detail.

Secondary electrons hit a scintillator which emits light which is converted into
electrical current and amplified. The signal is sent to a cathode ray tube and forms
image like the TV picture.

SEM was first introduced in 1965. It produces three-dimensional image, which has
high resolution and great depth of field.
Electron beam passes through a pair of scanning coils or deflector plates in the
electron column of the SEM in the final lens which deflects the beam on the surface
of the specimen. The electrons interact with atoms on the surface of the specimen
producing signals that contain information about the properties of the specimen.

The signals produced in an SEM include secondary electrons, back scattered

electrons (BSE). X-rays, transmitted electrons and light. The signals scan the surface
of the specimen from side to side, in lines from top to bottom. This is known as
roster scanning. They produce high resolution images of the specimen surface and
provide information about the composition of the specimen.

Phase Contrast Microscope:

Normally cells are studied after fixation and staining of the tissues. Only dead cells
can be studied in optical microscopes. But phase contrast microscope provide a
technique for studying transparent cells without staining. Different organelles
appear in various shades of grey depending upon the thickness and difference
between refractive index of the object and the medium. This microscope is most
suitable for studying cells cultured in vitro, during mitosis, protozoans, living cells
etc.

Phase contrast microscope is based upon the phase contrast principle of Fredrick
Zernike who developed phase contrast microscope called Zernike microscope in
1933. He was awarded Nobel Prize in 1953 for the discovery of phase contrast
principle.
If direct rays and diffracted rays of an object are in phase—having same amplitude
and frequency with each other, the resultant increased amplitude is doubled. It is
called constructive interference or coincidence. The object appears brighter than the
surroundings.

On the other hand if direct and diffracted rays are out of phase, it is called
destructive interference or interphase. The rays are partially cancelled and the object
appears dim and darker than the surroundings. Both these phenomena are used in
phase contrast microscope.

Zernike discovered that if light rays are diffracted differentially, they can cause
interference pattern in the image. Phase contrast microscope (PCM) has the ability
to separate the direct light or incident light from the light diffracted by the specimen.
Zernike developed a system of rings located both in the condenser lens and objective
lens.

The light rays which arrive at the eye are out of phase and the image of specimen
becomes enhanced. Colourless, transparent and invisible details of the specimen
become visible PCM enhances the contrast of the transparent and colourless objects
by influencing the optical path of light, causing difference in brightness. Invisible
objects can be seen. It is a contrast enhancing optical technique.
For this purpose, PCM has two optical attachments. The first one is a diaphragm (D),
which has an annular ring at the front focal plane of the condenser. It allows only a
ring of light to pass through. The second is a phase-shifting plate (P) at the rear focal
plane of the objective. It is transparent disc with an annular groove which coincides
exactly with the image of the diaphragm. All the direct light now passes through the
groove in the phase plate whereas the differenced light passes mainly outside the
groove of the phase plate.

If the phase plate is made to retard the incident wave by a quarter of wavelength
(1/4λ) the crest and though of the two waves will coincide, giving a resultant greater
amplitude. Refractive details will appear brighter or dark.

As the diffracted light has to pass through a greater thickness of transparent glass
material, a phase difference depending upon he refractive index of the phase place
and the groove is created between direct and diffracted light rays. This altering of
path of light rays is used to differentiate the different components of the cell or the
specimen.

This is because different components of the specimen cause light rays deviate
differentially or variably depending upon the density of each of them. It causes
difference in brightness which enables us to study the transparent and colourless
components. The different components of the specimen appear in various shades of
gray.

PCM has made it possible to study living cells without staining, study of cell division,
cell cycle, bacteria, sperms, tissues and sections etc.