Electron Microscopy Principle and Application

1. Electron Microscopy.
Principle, Source of illumination
and Application of TEM.[Transmission
electron microscopy is a major analytical method in the physical,
chemical and biological sciences].
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2. Learning Objectives
To understand concept of
Electrons used for Transmission.
The basic Principles of Recording
the Ultrastructures of living cells.
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3. Session Outcomes
• Learn the components of the Tool,
Principle and Application of TEM.
• Understand the significance of
Electrons in this process.
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4. Prerequisites
• Structure,Constituents and various
Components of cells is understood
and recorded on a led/tv screen by
TEM.[The image is then magnified and focused onto an imaging
device]. ,
• So basic defination of electrons used in
imaging cells and crystals and the
understanding of the term Transmission.
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5. Definition of Electron Microscopy.
Invention:-Ernst Ruska was awarded the 1986 Nobel Prize in
Physics “for his fundamental work in electron optics, and for the
design of the first electron microscope.” It is one of the most
influential scientific inventions, literally determining our view of the
nanoworld. George Palade later termed it the “birth certificate” of
cell biology.
• The smaller de Broglie wavelength of electrons enables the
instrument to capture fine detail—even as small as a single column of
atoms, which is thousands of times smaller than a resolvable object
seen in a light microscope. Also, capable of imaging at a significantly
higher resolution than light microscopes.,
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6. Electron Microscopy.
Scanning EM.
STEM[Scanning tunnelling electron micrography].
Transmission EM.
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7. The other types of similar microscopy are as follows ::
1)Environmental/In-situ TEM.
2)Aberration corrected TEM.
3)Ultrafast and dynamic TEM.
4)High resolution TEM.
5)Transmission Electron Aberration-
Corrected Microscope (TEAM)
6) Low-voltage elertron microscope.
Layout of optical Compo-
nents in a basic TEM.
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8. A Technical Introduction to Transmission Electron Microscopy for Soft‐
Matter: Imaging, Possibilities, Choices, and Technical Developments
The imaging method representation::
Small,
Volume:
16,
Issue:
14,
First
published:
04
March
2020,
DOI:
(10.1002/smll.201906198)
The make of an EM:: The
column is a stack of
electromagnetic lenses along
the optical axis of the
electron microscope .
underneath the electron
source there are two or
more condenser lenses,
which control the spot size
(C1) and the intensity (C2).
Below the condenser lenses
sits the objective lens. Most
TEMs have an objective
twin‐lens comprised of two
electromagnetic fields, one
above the sample, giving
extra control over the beam,
and one below the sample,
magnifying the image
roughly 50 times.Thus,
forming a diffracted Images.

9. Properties of Electrons.
● The atom contains negatively charged particles;with a high kinetic energy
revolving the nucleus of a atom:: Importantly these properties majorly involve in
imaging the living cells with high resolution.
● Electrons are optimal for studying soft materials at high magnifications, except for
two aspects that may constrain application:
● i) samples must be analyzed in vacuum and
● ii) possible radiation damage.
● Both issues demand measures to limit their effect and are recurring themes in
microscope design, sample preparation, imaging and image processing.
Single crystal LaB6 filament.
9

10. Principle of Transmission Electron
Microscopy.
A TEM image is formed by propagating a bundle of waves (beam) onto a sample. Some of the
waves interact with the sample after which the resulting image is magnified by a series of
lenses. There are two types of interactions between electrons from the beam (primary
electrons) and atoms from the sample: elastic and inelastic scattering::
In an event of elastic scattering, the combined
kinetic energy (E = mv2) and the combined
momentum (E = mv) of the atom and the
interacting electron is the same before and
after their interaction.
Inelastic scattering occurs when the amount of
kinetic energy or momentum is not conserved.
scattering processes are less localized due to
electron-electron interactions.
The high resolution-- information from the
sample is present in the elastically scattered
electron. The Difference in an Electron and a
nucleus has less momentum to the nuclues.,
hence all the scattering events are essentially
elastic.
To increase the total number of scattering
events and change the ratio between elastic
and inelastic scattering, heavy metals can be
added to samples that contain mainly carbon
such as most soft materials. This is typically
referred to as staining.
Electron Sources
There are two types of electron sources: thermionic and field‐emission guns (FEGs).
1)::The slow‐scan charge‐coupled device (CCD) camera.
2)::DDD cameras are capable of directly detecting electrons, avoiding blurring and improving
recording spee
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11. Specimen Preparation.
Ultra thin sectioning / ion
etching.,
Metal Shadow casting with
Au/Ag.,--Mechanical milling
.
Freeze etching or
cryp-preservation.
Tissue sectioning/Drying
the Sample.
Cryo –Fixation: is a
perservation method.
needs to balance the choices of
contrast (addition
: A diamond, or cubic boron A
diamond or cubic boron nitrides
meSputtering with argon gas
generates plasma stream.al
milling.
AA A diamond or
achievable resolution and native
state versus more artificial
preservation.
CryoSt Simple Staining
with Osmium tetra oxide
nc
Sample Staining
/negative staining ..
Au-- gold (in immunogold labelling) A
diamond or cubic boron nitrides are
used to polish in mechanical milling.
Tissue sectioning/Drying
the Sample.
needs to balance the
choices of contrast (addition
uses N2- di -oxide./
liquid N2.olution and
native state versus more
artificial preservation.
tiveuses nigrosine or
indian ink.
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14. Application of TEM.
• Transmission electron microscopy is
used to reveal sub-micrometre, internal
fine structure in solids.
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15. Biologically Important Tool.
Materials scientists tend to call the study of crystals having
microstructure while bioscientists usually prefer the term
ultrastructure for imaging and recording the living cells by the use
of TEM..
15

16. Recap
● Use of Electrons
● Expand TEM.
● Principle
● Preparation of Specimen.
● Applications
● Precautions:---Without precautions, cells, membranes, aggregates and artificial systems
will all be deformed and rearranged, and molecular concentrations will be altered. 16

17. Questions
2. why the electron microscope is able to offer higher resolution than
the light microscope.
3. EM uses the accelerated beams obtained from the ______ in the
______.
4. Mention the types of abberations used in TEM.
5. The Sperical abberation is reduced by ______ _______ in TEM
● Which of the following techniques are used in Transmission Electron
Microscopy (TEM) for examining cellular structure?
● A Negative-Staining
● B Shadow Casting
● C Ultrathin Sectioning
● D All of the above.
●
1. Both light microscopes and TEMs form images on __________.
17

18. 2 MCQs.
1. Why TEM images have much higher resolution than images from light
microscopes?
A. TEM is much greater in size than light microscope .
B. Electrons traveling as waves have wavelengths much shorter than
visible light .
C. TEM can achieve greater magnification .
D. The fluorescent screen of TEM can generate high resolution images.,
2. With the use of ultramicrotome, we can section our sample into
thickness of…
A. 1 nm
B. 10 nm
C. 100 nm
D. 1000 nm.,
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19. 3. MCQs.
7. Which of the following is the correct pathway of electrons in the
TEM?
A. Anode → electromagnetic lens system → sample → fluorescent
screen
B. Anode → electromagnetic lens system → sample → electromagnetic
lens system → fluorescent screen
C. Cathode → electromagnetic lens system → sample →
electromagnetic lens system → fluorescent screen
D. Cathode → electromagnetic lens system → sample → fluorescent
screen.
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20. 5.. For viewing which of the following would a TEM not be a good
choice?
A.Antigen
B. Actin filament
C. 3D external surface of mitochondria
D. Ribosomes
6. Degree of scattering in transmission electron microscope is a
function of __________
a) wavelength of electron beam used
b) number of atoms that lie in the electron path
c) number and mass of atoms that lie in the electron path
d) mass of atoms that lie in the electron path
View Answer
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21. References
• At the bench a laboratory navigator --- by Kathy becker.,
• Microbiology by Pelczar ::Microbiology Pelczar 6th Edition.,
• A laboratory manule --- Experiments in Microbiology, Plant Pathology
and Biotechnology by K.R.Aneja.,
• Microbiology ---text book by Chandrakanth kelamani.,
• General Microbiology (Vol 1) | Powar & Daginawala.,
• A Textbook of Microbiology – by P CHAKRABORTY MICROBIOLOGY..
• EBOOK LIBRARY
• Microbiology Pelczar 6th Edition PDF, ePub eBook.,
• Library resources about::--Transmission electron microscopy.,
• The Wikibook Nanotechnology has a page on the topic of: Transmission
electron microscopy (TEM)
• https://www.slideshare.net/suniu/electron-microscopy.,
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