The document discusses the principles and workings of transmission electron microscopes (TEM). Key points: - TEMs use electron beams instead of light to achieve much higher magnifications, allowing observation of objects as small as 0.2 nm. - Electrons are emitted from a heated filament and accelerated through magnetic lenses, which focus the beam onto ultra-thin specimen sections. - Interactions between electrons and the specimen create an image that is magnified and detected, allowing visualization of internal structures at high resolution. - Proper sample preparation including fixation, dehydration and thin sectioning is crucial for TEM to work, as it requires specimens thin enough to be transparent to electrons.

1. PAPER 4
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PRINCIPLE OF TRANSMISSION
ELECTRON MICROSCOPE

2. INTRODUCTION
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 Electron microscopes are scientific instruments
that use a beam of energetic electrons to
examine objects on a very fine scale.
 It has greater magnification than light
microscope and hence we can see things that we
would not normally be able to see with our naked
eyes.
 10,000X plus magnification, not possible using
current optical microscopes.

3. TRANSMISSION ELECTRON
MICROSCOPE (TEM)
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 TEM was the first type of electron microscope to
be developed and it is patterned exactly on the
light microscope except for it uses a focused
beam of electrons instead of light to see through
the specimen.
 Developed by Max Knoll and Ernst Ruska,
Germany, 1931.
MICROSCOPE RESOLUTION MAGNIFICATION
OPTICAL 200 nm 1000X
TEM 0.2 nm 5,00000X

4. TEM
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 It is a microscopy technique whereby a beam of
electrons is transmitted through an ultra thin
specimen, interacting with the specimen as it
passes through. An image is formed from the
interaction of electrons transmitted through the
specimen; the image is magnified and focuses
onto an imaging device, such as a fluorescent
screen, on a layer of photographic film, or to be
detected by a sensor such as a CCD camera.

5. REPRESENTATION OF TEM’S
WORKING
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6. JEM-1010 TEM
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8. TEM
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 Source of illumination : a beam of electrons of
very short wavelength emitted from a tungsten
filament.
Optical system is enclosed in vacuum so as to
avoid collision with air molecules and scattering of
electrons.
 Tungsten filament : heated filament emits
electrons that are accelerated by the voltage in
the anode.
higher anode voltage higher electron speed
shorter
wavelength increased resolution.

9. TEM
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 Magnetic columns : placed at specific intervals
in the column. acts as
electromagnetic condenser lens system and
focuses the electron beam.
 Specimen : stained with electron dense material.
placed in vacuum.
electron beams pass through the specimen and
are scattered
by internal structures.

10. TEM
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 Transmitted beam : carries information regarding
the structure of the specimen.
 Magnetic lenses : magnify the spatial variation in
the information (image).
 Image recorder : fluorescent screen,
photographic plate, light sensitive sensor like
CCD camera.
 Image display : real time on a monitor or
computer.

11. ELECTRON GUN
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• The function of an electron gun is to emit an
intense beam of electrons into the vacuum
which accelerates the between the cathode and
the anode. The metals contain free
electrons. The valence are free electrons, which
are loosely bound in the nucleus.
Those electrons cannot escape from the metal
surface . The positively charged nucleus will try to
pull back the free electrons when they try to
escape from the surface. Hence the electrons
have to overcome the potential barrier in order
to escape from the surface of the metals. The
energy required to overcome this potential barrier

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 Work function, φ, is the minimum energy in
electron volts required to remove an electron from
the metal surface. If the electrons in metals are to
be emitted from the cathode they have to
overcome the work function.
Electrons are emitted from a metal by two
methods:
 Thermionic emission: In this method the
electrons are emitted from the metals by heating
them.
 Field emission: In this method the electrons are
emitted from metals, under strong electric fields.

13. SAMPLE PREPARATION
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 Sample preparation is important for electron
microscopy. There are three main steps for
sample preparation: Processing, embedding
and polymerization.
Processing
This includes: fixation, rinsing, post fixation,
dehydration and infiltration.
 1) Fixation : This is done to preserve the sample
and to prevent further deterioration so that it
appears as close as possible to the living state,
although it is dead now.
 Eg. Gluteraldehyde fixation for proteins.

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 2) Rinsing : The samples should be washed with
a buffer to maintain the pH. This prevents extra
acidity.
 3) Post fixation : A secondary fixation with
osmium tetroxide (OsO4), which is to increase
the stability and contrast of fine structure.
 4) Dehydration : The water content in the tissue
sample should be replaced with an organic
solvent since the epoxy resin used in infiltration
and embedding step are not miscible with water.

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 5) Infiltration : Epoxy resin is used to infiltrate
the cells. It penetrates the cells and fills the space
to give hard plastic material which will tolerate the
pressure of cutting.
Embedding
After processing the next step is embedding. This is
done using flat molds.
Polymerization
Next is polymerization step in which the resin is
allowed to set overnight at a temperature of 60 degree
in an oven.

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 Sectioning : The specimen must be cut into
very thin sections for electron microscopy so that
the electrons are semitransparent to electrons.

17. OTHER METHODS OF SAMPLE
PREPARATION
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 1. CRYOFIXATION : Like chemical fixatives, it
stops the metabolic processes and preserves
biological structure.
The method involves ultra rapid cooling of
small samples to liquid nitrogen temperature (-
196°C) or below thus stopping all motion and
metabolic activity, and preserving the internal
structure by freezing all fluid phases solid.

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 2. NEGATIVE STAINING : The main purpose of
negative staining is to surround or embed the
biological object in a suitable electron dense
material which provides high contrast and good
preservation.
This method is capable of providing information
about structural details often finer than those
visible in thin sections, replicas or shadowed
specimens.
In addition to the possibility of obtaining a
spectacular enchantment of contrast, it has
advantage of speed and simplicity.

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 3. SHADOW CASTING : The grid containing the
specimen are placed in sealed chamber which is
evacuated by vacuum pump.
The chamber contains a filament composed of a
heavy metal together with carbon.

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 4. FREEZE FRACTURE REPLICATION AND
FREEZE ETCHING : Small pieces of tissues are
placed on a small metal disk and rapidly frozen.
The disk is then mounted on a cooled stage
within a vacuum chamber and a frozen tissue block
is struck by a knife edge.
The resulting fracture plane spreads out from the
point of contact, splitting the tissue into two pieces.

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23. TEM IMAGE OF T4
BACTERIOPHAGE ATTACKING
HOST
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24. TEM IMAGE OF S. aureus
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25. TEM PLATINUM REPLICA IMAGE
OF S.aureus
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26. TEM IMAGE OF POLIO VIRUS
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27. CONCLUSION
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 Like in any multistep preparation procedure,
virtually every step can affect the quality of the
final electron micrograph.
 A single mistake in one of these steps will affect
all the remaining steps, and thus the outcome of
the entire study.
 Most of the chemicals used in these procedures
are chemically dangerous and potentially
hazardous.
 These procedures are time consuming and
require skills.

28. REFERENCES
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 Wikipedia
 Google images
 Research Gate

29. THANKYOU!
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