Electron Microscope working principals and its various aspects in pathology

1. ELECTRON MICROSCOPE
Dr AKHIL S
Malabar Dental Collge
DEPARTMENT OF ORAL PATHOLOGY
SEM
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2. The Central player - (e)
• The electron “e” is an elementary particle
• Also called corpuscle
• carries a negative charge.
• the electron was discovered by J. J. Thompson in 1897
• e is a constituent of the atom
• 1000 times smaller than a hydrogen atom.
• the mass of the electron 1/1836 of that of a proton.
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3. Scheme of electron-matter
interactions arising
from the impact of an electron beam
onto a specimen.
A signal below the specimen is
observable if the
thickness is small enough to allow
some electrons to
pass through
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4. Elastic Electron Interactions
• no energy is transferred from the electron to the
sample.
• These signals are mainly exploited in
- Transmission Electron Microscopy and
- Electron diffraction methods.
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5. Inelastic Electron Interactions
- Energy is transferred from the electrons to the specimen
- The energy transferred can cause different signals such as
- X-rays,
- Auger electrons
- secondary electrons,
- plasmons,
- phonons,
- UV quanta or cathodoluminescence.
• Used in Analytical Electron Microscopy … SEM
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6. What is Electron Microscopy?
• Electron microscopy is a diagnostic tool with
diversified combination of techniques ……
• that offer unique possibilities to gain insights into
- structure,
- topology,
- morphology, and
- composition of a material.
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7. What is an Electron Microscope ?
• A special type of microscope having a high resolution of images, able
to magnify objects in nanometres, which are formed by controlled
use of electrons in vacuum captured on a phosphorescent screen
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8. Why were the EMs invented?
• To study objects of < 0.2 micrometer
• For analysis of sub cellular structures
• Intra cellular pathogens - viruses
• Cell metabolism
• Study of minute structures in the nature
Greater resolving power of the EMs than
light microscope
• An EM can magnify structures from 100
– 250000 times than light microscopy
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9. The novelty of EMs from others
• Beam of Electrons …… instead of a beam of light
• Electro-magnetic lens ………..instead of Ground glass
lenses
• Cylindrical Vacuum column - Electrons should travel
in vacuum to avoid collisions with air molecules that
cause scattering of electrons distorting the image
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10. 10

11. Types of electron microscope
1. Transmission electron
microscopy :
2. Scanning electron
microscopy:
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12. SEM
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13. Transmission Electron Microscope
Principle
• TEM is the direct counterpart of Light microscope
• Involves passage of high velocity electron beam
through specimen, thin enough to transmit 50% of
the electrons
• Transmitted electrons – focused by lens systems to
form a 2 dimensional magnified image
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14. Analogy between LM & TEM
• Arrangement & function of their components
1. Illuminating system – source & condenser
2. Imaging system – lenses to produce magnified
image – objective & projector
3. Image translating system – Final image is viewed
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15. THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE
VacuumAir-filledInterior
MagnetsGlassLenses
High voltage (50kV)
tungsten filament
Tungsten or quartz
halogen lamp
Radiation
source
X 5,00,000x1000 – x1500
Maximum
magnification
0.14nm
Fine detail
app. 200nm or
0.2micron
Maximum
resolving power
Electrons
app. 4nm
Visible light
390nm (red) – 760nm
Electromagnetic
spectrum used
ELECTRON MICROSCOPELIGHT MICROSCOPEFEATURE
© 2007 Paul Billiet ODWS
Focus Lens is movable
Rigidly fixed, adjust
lens currents
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16. THE LIGHT MICROSCOPE v THE ELECTRON
MICROSCOPE
Copper gridGlass slideSupport
Heavy metalsWater soluble dyesStains
Ultramicrotome
Slices - 50nm
Parts of cells visible
Microtome
slices - 20 000nm
Whole cells visible
Sectioning
ResinWaxEmbedding
Glutaraldehyde,OsO4formaldehydeFixation
ELECTRON
MICROSCOPE
LIGHT MICROSCOPEFEATURE
© 2007 Paul Billiet ODWS
Focussing screen
Human eye (retina),
photographic film
Fluorescent screen,
photographic film
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17. PARTS IN DETAIL
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18. Electron source
• The electron source consists of a
cathode and an anode.
• The cathode is a tungsten filament
which emits electrons when being
heated.
• A negative cap confines the
electrons into a loosely focused
beam.
• The beam is then accelerated
towards the specimen by the
positive anode.
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19. Electromagnetic lens system
• The system allows electrons within a small
energy range to pass through, so the electrons
in the electron beam will have a well-defined
energy.
• 1. Magnetic Lens: Circular electro-
magnets capable of generating a precise
circular magnetic field. The field acts like an
optical lens to focus the electrons.
• 2. Aperture: A thin disk with a small (2-100
micrometers) circular through-hole. It is used to
restrict the electron beam and filter out
unwanted electrons before hitting the
specimen.
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20. The Vacuum System
• The electron beam must be generated in and traverse
through the microscope column under a high vacuum
condition.
• The presence of air molecules will result in the
collision and scattering of the electrons from their
path.
• In the electron microscope the vacuum is maintained
by a series of highly efficient vacuum pumps.
• THE VACUUM FACTOR: Biological material must be
properly fixed and preserved 20

21. Sample holder
• The sample holder is a platform equipped with a
mechanical arm for holding the specimen and
controlling its position.
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22. Imaging system
• The imaging system consists of
another electromagnetic lens system
and a screen.
• The electromagnetic lens - two lens,
one for refocusing the electrons after
they pass through the specimen, and
the other for enlarging the image and
projecting it onto the screen.
• The screen has a phosphorescent plate
which glows when being hit by
electrons.
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23. Image Formation in the TEM
• The basis of image formation in the TEM is the scattering
of electrons.
• The scattering results in a shadow on the viewing screen
or photographic film.
• Material with high atomic numbers will cause more
scattering and produce a deep shadow. Such material is
termed "electron dense" and has high image contrast.
• Biological material has low electron density and is known
generally as "electron transparent". Hence, an inherent
low contrast image is formed.
• BIOLOGICAL MATERIAL must, therefore, be STAINED with
heavy metal salts.
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24. • ELECTRON SOURCE
• ELECTROMAGNETIC LENS
SYSTEM
• SAMPLE HOLDER
• IMAGING SYSTEM.
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25. THE SCANNING ELECTRON MICROSCOPE
• To directly visualize the surface topography of solid
unsectioned specimens.
• Probe scans the specimen in square raster pattern.
• The first scanning electron microscope (SEM) debuted in
1938 ( Von Ardenne) with the first commercial
instruments around 1965.
• Differs from TEM in construction & operational modes
• TEM – information is obtained from transmitted electrons
• SEM – majority is obtained from secondary,
backscattered electrons & from X-rays.
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27. 27

28. 28

29. • After the impingement of the primary
electrons on the specimens, secondary
electrons as well as other forms of radiation
are emitted.
• But only the secondary electrons will be
collected by the signal detector.
• In the detector these electrons strike a
scintillator and the light produced is
converted to electric signals by a
photomultiplier.
• The electric signal is then amplified and
displayed on the cathode ray tube (CRT).
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30. • In the SEM the electron beam is rapidly scanned back
and forth in an orderly pattern across the specimen
surface.
• It is a composite of many individual image spots
similar to the image formed on the TV screen.
• The SEM has a specimen stage that allows the
specimen to move freely so that the surface of the
specimen can be viewed from all angles.
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31. The focused electron beam is moved from one pixel to another.
At every pixel, the beam stays for a defined time and generates
a signal (e.g.secondary electrons) which are detected, amplified
and displayed on a computer screen
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32. • A smaller area is scanned with the same number of pixels.
• The scanned pixels are smaller
Image magnification in SEM
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34. TEM vs SEM
TEM SEM
5 lenses – C1, C2, objective, 2
projector
3 lenses – 2 condensor, 1
objective
High accelerating voltage -
penetration
low accelerating voltage
Not complicated Specimen Stage – complicated
X & y axis X,Y,Z-axis, tilting, rotating
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35. • Diseases of kidney
• Metabolic storage diseases
• Respiratory tract biopsies
• Skeletal muscle diseases
• Infectious agents
• Cutaneous diseases
• Peripheral nerve biopsies
• Epithelial tumors
• Mesothelioma
• Melanoma
• Hematopoietic and
lymphopoietic tumors
• Soft-tissue tumors
• Central nervous system
tumors
• Small round cell tumors
Non tumor biopsies Tumor diagnosis
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36. A, Adenovirus, an icosahedral nonenveloped DNA virus with fibers. B, Epstein-Barr virus, an
icosahedral enveloped DNA virus. C, Rotavirus, a nonenveloped, wheel-like, RNA virus. D,
Paramyxovirus, a spherical enveloped RNA virus. RNA is seen spilling out of the disrupted
virus
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37. Squamous cell carcinoma
Well differentiated squamous cell carcinoma
Abundant cytokeratin filaments.
Frequent desmosomes between cells.
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38. Well differentiated squamous cell
carcinoma- frequent desmosomes 38

39. Disadvantages
1. EM is not economical- stable high voltage supply,
vaccum system etc
2. Findings unlikely to influence treatment, IHC & LM
together are confirmatory .
3. Tissue preparation is tough
4. Only a small proportion of neoplasm can be studied
5. Misinterpretation of non- neoplastic elements
belonging to the tumor
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40. Conclusion
Currently the use of EM is limited for the
expense and lack of surgical pathologists to
interpret EM findings .
Still it provides unique insight into the
structure of some tumors and renal
pathologies.
So better to use it selectively in study and
diagnosis of human diseases and research areas
and correlating the findings with LM findings
and IHC results.
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41. References
• Theory and practices of histopathological techniques,
John D Bancroft, 4th edition;.
• Robbins and Cotran, PATHOLOGIC BASIS OF DISEASE, 8th
edition.
• Cellular pathology technique, C.F.A. Culling, 4th
edition;pg .
• Gail Stewart .Microscopes. Kidhaven Press,1 2002:23=65.
• Shar Levine, Leslie Johnstone The Ultimate Guide to Your
Microscope. Sterling Publishing Company, Inc.,3 2008:
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42. • Kenneth Rainis & Bruce Russell Guide to Micro life .Microscope and
micro life. Watts 2 2001:11-76.
• Elizabeth M. Slayter .Light and Electron Microscopy. Cambridge
University Press, 1: 30-Oct-1992.
• Various internet sources.
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