2. Electron Microscope
Microscopes that are use electrons as the light source and electromagnetic
coils to direct the path of the e- are called as electron microscopes.
(The optical system is completely replaced by electromagnetic coils).
The first electron microscope was designed by Knoll and Ruska (1931).
(Wavelength of e- = 0.05A very short wavelength with very high
magnification).
The magnification of electron microscope is 1000 times higher than the
light microscope. (Therefore the magnification of e- is 100 × 1000 = 1,
00,000 X.)
3. Types of Electron Microscope
Transmission electron microscope ( TEM )
Scanning electron microscope ( SEM)
4. Transmission electron microscope
(TEM):
In this type e- are allowed to transmit through the
specimen is called TEM.
The first TEM was designed by Max Knoll and Ernst
Ruska (1931).
6. Principle
The basic principle of electron microscope is similar to the compound
microscopes but the e- beam is substituted for light source and
electromagnetic coils to optical lens.
When high voltage current is passed through the cathode ray tube,
e- beams are produced.
Electromagnetic coils direct the e- beams to pass through the
specimen.
It is stained with gold or osmium and the image is collected by
objective lens and amplifier (electromagnetic coils).
The image cannot be seen by our naked eye, so it is casted on a
screen or photographic plate or camera.
7. Electron microscopes are kept in
vacuum because
1. Electrons are easily absorbed in air.
2. Electrons are move in a straight line
only in vacuum.
8. Instrumentation: The TEM has an
electron gun and condenser lens.
A) Electron gun
B) Condenser lens
C) Objective lens
D) Amplifier lens
E) Projector lens
F) Ancillary equipment
9.
10. A) Electron gun:
Made up of cathode ray tube with tungsten filament
(2mm long)
Located at the top of the microscope.
It generates e-
11. B) Condenser lens
Two condenser lens or electromagnetic coils are present below the e-
gun.
They collect and direct the beams into the specimens on a stage.
A thin section of specimen is placed on a thin plastic film mounted on
a copper gird (3 mm diameter).
12. C) Objective lens
It is an electromagnetic coil placed below the
specimen stage.
It collects the specimen image and focus towards
the amplifier lens.
13. D) Amplifier lens
It is an electromagnetic coil below the objective
lens and magnifies the image several times.
15. F)Ancillary equipment
1. The entire set up is placed in a vacuum
tube.
2. TEM release large amount of heat during
working hours, so cooling system is present
3. It needs high power supply
16. Preparation of specimen for TEM:
Biological material contains low atomic weight elements like carbon,
hydrogen, oxygen and nitrogen.
They do not give high resolution.
Therefore, the biological sample has to be loaded with heavy atoms like
gold or osmium and these atoms protect the specimen from destruction.
17. It involves the following steps.
The specimens are dehydrated by keep it in
ethanol or acetone.
Place it chemical fixative like Osmium tetra
oxide. (These fixatives form covalent bonds
with biological molecules).
After fixation, the specimen is embedded in
araldite or plastic medium.
18. It involves the following steps
The embedded specimen is cut in to thin sections
of 50-100nm thickness using a glass or diamond
knife in an ultra microtome.
The thin sections are mounted on the copper
grid.
The grid with specimen is mounted on the
specimen stage.
The image is viewed on fluorescence screen.
20. Applications
TEM is an ideal tool for the study of ultra
structure of a cell.
It is used to identify plant and animal virus.
It is widely applied in various researches in
oncology, pollution, biochemistry, molecular
biology, etc.
21. Disadvantages:
Very high cost.
We cannot study 3 dimensional structures of the
specimens.
The specimens should be fixed properly and
should take ultra thin sections, because an
electron has limited penetrating power.
We could not study live specimens.
It is successful only under high vacuum
condition.
23. Scanning Electron Microscope
In SEM, the surface of the specimen is
scan by electron beam.
This was first designed by Max Knoll
(1935).
24.
25. Principle:
SEM use electron beam for illumination and electromagnetic coils for
directing the path of e- beam.
When e- is focused on the specimen, it produces secondary e- (SE), back
scattered e- (BSE) and characteristic X-rays.
Secondary electrons are reflected due to the interactions between atoms
in specimens and e- beam.
Back scattered e- gives information about the distribution of different
elements.
26. Characteristic X-rays are emitted by the sample when the e- beam removes
e- from the inner shells of the atoms of the specimens.
These three rays are detected by specialized detectors.
Electronic amplifier like collectors, Scintillator and Photomultiplier
(PMT) are used to measure the e- signals.
The e- signals are converted in to image which is focused on the monitor.
27.
28.
29. Instrumentation:
Electron gun:
it is the source of e- beam and located
at the top of the microscope.
It consists of cathode plate and anode
plate.
30. Condenser lens:
there are two condenser lenses just below the
e- gun.
They collect and concentrate the e- in to a
strong beam.
31. C.Deflection coils:
below the condensers, there is a
deflection coil to direct the beam of e- in
to the specimen stage.
32. D. Specimen stage:
it is present in slanting position at the
lower side of deflection coil.
33. E.Separate e- detectors
( scintillator& PMT ) are attached in the
vacuum tube.
Electronic amplifiers are connected with
detectors.
The electric signals are converted into bright
spots of varying density by scanning circuit.
34. Additional things
G. Image is displayed on a photographic plate
or computer monitor.
H. The entire set up should be placed in a
vacuum tube.
I. Power supply with high voltage.
J. SEM releases huge amount of heat, so
cooling system is present around it.
35. Preparations of specimens for SEM:
Dry materials like wood, bone, feathers,
insect’s wings and shells are coated with thin
film of electro conductive materials like
gold, platinum, tungsten, osmium, chromium
and graphite.
Then the specimens are placed on the stage.
36. Preparation of wet specimens involves
the following steps.
Specimens are fixed by fixatives like osmium tetroxide, potassium
permanganate, formalin,etc. which stabilize molecular organization of
specimens.
They are dehydrated by keeping in the increasing concentrations of ethanol
or acetone.
The specimens are coated with ultra thin layer of electro conductive alloy.
Then they are put in the specimen stage.
The e- beam is passing through the specimen and final image is produced
on the computer screen.
39. Advantages:
SEM is use full to view the surface of
microorganisms (Bacteria, Diatoms), pollen grains,
hairs and scales of plants and animals.
It is free of chromatic aberrations.
It produce 3D image.
SEM is used study archeological specimens and
fossils.
It is used to analyze the compound eyes of
insects.
40. Disadvantages:
Lower resolution than TEM.
High cost.
Complete vacuum is needed.
Factors limit the quality is
uncontrolled emission of e- and scan
faults.