ELECTRON MICROSCOPY

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 ELECTRON MICROSCOPE
• Electron microscope work by using an
electron beam instead of visible light & an
electron detector instead of our eyes & the
magnification is obtained by
electromagnetic field .
• Electron beam has the properties of a wave
with a wavelength that is much smaller than
visible light .
• The smaller is the wavelength of light , the
greater is the resolving power .
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• Electron microscope examine objects on a very
fine scale & this examination can yield
information about the topography , morphology ,
composition & crystallographic information .
• The electron microscope is best used for
studying biological ultra-structure & the image
obtained is called electron micrograph .

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 HISTORY

• It was Ernst Ruska & Max Knoll , a physicist

& an electrical engineer , respectively from
the University of Berlin , who created the first
electron microscope in 1931 .
• The first commercial electron microscope
was produced in 1938 by Siemens .
• Albert Prebus & Siemens produced a
transmission electron microscope (TEM) in
1939 .

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 RESOLUTION & MAGNIFICATION

• resolving power of an electron microscope is 200

times greater than that of a light microscope .
• It produces useful magnification up to 400,000 X
as compared to 2000X in a light microscope .

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TYPES OF ELECTRON MICROSCOPE
• There are three types of electron microscope
as described below :-
1) Transmission electron microscope (TEM)
2) Scanning electron microscope (SEM)
3) Scanning transmission electron microscope
(STEM)

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1) TRANSMISSION ELECTRON
MICROSCOPE (TEM)

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Structural Parts of TEM

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 Principle & Working of TEM
• Specimen is bombarded by a beam of electrons ,
the primary electrons . The bombarding
electrons are focused onto the object .
• In areas in the object where these electrons
encounter atoms with a heavy atomic nucleus ,
they rebound .
• In regions where the material consist of lighter
atoms , the electrons are able to pass through .
• The fine pattern of electrons leaving the object ,
reaches the objective lens forms the image .
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• It is then greatly enlarged by projector lens .
• Eventually , the tranversing electrons
(transmission) reach the scintillator plate at
the base of the column of the microscope .
• The scintillator contains phosphor
compounds that can absorb the energy of
the stricking electrons & convert it to light
flashes .
• Thus , a contrasted image is formed on this
plate .

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 Sample Preparation for TEM
• The material to be studied under electron
microscope must be well preserved , fixed ,
completely dehydrated , ultrathin & impregnated
with heavy metals that sharpen the difference
among various organelles .
• The material is preserved by fixation with
glutaraldehyde & then with osmium tetroxide .
• The fixed material is dehydrated & then embedded
in plastic (epoxy resin) & sectioned with the help
of diamond or glass razor of ultra- microtome .
• The sections are ultrathin about 50-100 nm thick .

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• These sections are placed on a copper grid &
exposed to electron dense materials like lead
acetate , uranylacetate , palladium vapours ,
phosphotungstate etc . Now the sections can
be viewed in the TEM .
• The coating with electrons dense materials
enables the specimen to withstand electric
bombardment .

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 Application of Ttransmission
Electron Microscopy

● To study structures of bacteria, fungi, and

viruses
● To study shape and size of microbial cell
organelles
● To study bacterial flagella and plasmids
● Differentiate pland cell and animal cells
● To study about nano particles

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 Advantages of TEM
• TEMs offer very powerful magnification &
resolution .
• TEMs provide information on element &
compound structure .
• Images are high – quality & detailed .

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 Disadvantages of TEM
• TEMs are large & expensive .
• Laborious sample preparation .
• Operation & analysis requires special training .
• Samples are limited to those that are electron
transparent .
• TEMs require special housing & maintainance .
• Images are black & white .
• Live specimens cannot be observed
• Specimen should be ultra thin

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2) SCANNING ELECTRON
MICROSCOPE (SEM)

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Structural Parts of SEM

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 Principle & Working of SEM
• The electron gun produces an electron beam when
tungsten wire is heated by current .
• This beam is accelerated by the anode .
• The beam travels through electromagnetic fields &
lenses , which focus the beam down toward the
sample .
• A mechanism of deflection coils enables to guide
the beam so that it scans the surface of the sample
in a rectangular frame .
• When the beam touches the surface of the sample ,
it produces :
- Secondary electrons (SE)
- Back scattered electrons (BSE)
- X- Rays …
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• The emitted SE is collected by SED & convert it into
signal that is sent to a screen which produces the final
image .
• These signals are scanned in the manner of a
television system to produce an image on a cathode
ray tube (CRT) .
• The image is recorded by capturing it from the CRT .
• Additional detectors collects the X-rays , BSE &
produce corresponding images .
• A secondary electron detector (SED) attracts the SE &
depending on the number of electrons that reach the
detector , registers different levels of brightness on a
monitor .

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 Sample Preparation for SEM
• The specimen is first fixed in liquid propane then
dehydrated in alcohol
• The dried specimen is the coated with a thin film
of heavy metal , such as platinum or gold , by
evaporation in a vacuum provides a reflecting
surface of electrons .
• The surface of the specimen when scanned by the
electron beam release secondary electrons that
form a three-dimensional image of the specimen
on a television screen .
• Holes & fissures appear dark , & knobs & ridges
appear light .
• Complete scanning from top to bottom usually
takes only a few seconds
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 Advantages of SEM
• It gives detailed 3D & topographical imaging &
the versatile information generated from
different detectors .
• Modern SEMs allow for the generation of data
in digital form .
• Most SEM samples require minimal preparation
actions .

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 Disdavantages of SEM
• SEMs are expensive & large .
• Special training is required to operate an SEM .
• SEMs carry a small risk of radiation exposure
associated with the electrons that scatter from
beneath the sample surface .

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 3) SCANNING TRANSMISSION
ELECTRON MICROSCOPE (STEM)

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• A scanning transmission electron microscope
or STEM combines the capabilities of both a
SEM & a TEM.
• The electron beam is transmitted across the
sample to create an image (TEM) while it also
scans a small region on the sample (SEM) .

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 APPLICATIONS OF ELECTRON
MICROSCOPE
• Electron microscope is being used today
in research laboratories around the
world to explore the molecular
mechanisms of disease , to visualize the
3D structure of tissues & cells .
• Forensic science uses electron
microscopy to analyze criminal evidence
such as gunshot residue , clothing fibres ,
soil samples etc.
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• Inorganic particles – both natural & manmade
including soil , coal , cement , fly ash etc . can
be analyzed to provide more detailed
understanding of impact of waste pollution
on environment & health .
• In medical field electron microscope is used
to compare healthy & unhealthy blood &
tissue samples .

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