The document discusses electron microscopy techniques. It provides an overview of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM uses a beam of electrons to produce images of sample surfaces, while TEM transmits electrons through thin samples to form magnified images. The document outlines the basic components, working principles, and applications of SEM and TEM, such as viewing cell structures and analyzing material properties at high resolutions. Limitations include high costs, specialized training and sample preparation requirements.

1. ELECTRON MICROSCOPY
-SCANNING ELECTRON MICROSCOPE
-TRANSMISSION ELECTRON
MICROSCOPE
PRESENTED BY – SUMER PANKAJ
CLASS – MSC. EST
ROLL NO. - 42
Institute of Science and Technology for Advanced Studies and Research
Affiliated to Sardar Patel University Recognized under section 2(f) and 12 (B) of UGC
act 1956 Mota Bazaar, Vallabh Vidyanagar, Anand, Gujarat 38812058
PG Department of Environmental Science and Technology
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2. CONTENT
• Microscopy and its types
• Principle of electron microscopy
• Types of electron microscopy
• SEM vs. Tem
• Recent advancements in SEM &
TEM
• References
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SEM (Scanning Electron Microscope)
•Principle
•Components
•Instrumentation
•Working
•Applications
•Limitations
TEM (Transmission Electron Microscope)
•Principle
•Components
•Instrumentation
•Working
•Checklist
•Applications
•Limitations

3. INTRODUCTION
• Microscopy is the technical field of
using microscopes to view objects that cannot be
seen with the naked eye. There are three well-known
branches of microscopy: optical, electron and
scanning probe microscope.
• Optical and electron microscopy involve
the diffraction, reflection,
or refraction of electromagnetic radiation or electron
beams interacting with the specimen, and the
collection of the scattered radiation or another signal
in order to create an image.
• Scanning probe microscopy involves the interaction
of a scanning probe with the surface of the object of
interest.
1..
2.
3.
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4. Types of
Microscopy
Others
Optical
Microsco
py
Scanning
Probe
Microsco
py
Electron
Microsco
py
• Bright field
• Oblique
illumination
• Dark field
• Dispersion
staining
• Phase contrast
• Differential
interference
contrast
• Interference
reflection
• Fluorescence
• Confocal
• Wide-field
multiphoton
• Transmission
electron
microscopy
(TEM)
• Scanning
electron
microscopy
(SEM)
• Atomic force
microscope (AF
M)
• Scanning tunnel
microscope
• Photonic force
microscope
• Recurrence
tracking
microscope
• Ultraviolet
microscopy
• Infrared
microscopy
• Digital holographic
microscopy
• Digital pathology
(virtual
microscopy)
• Laser microscopy
• Amateur
microscopy
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5. PRINCIPLE OF ELECTRON
MICROSCOPE• An electron microscope is a microscope that uses a beam of
accelerated electrons as a source of illumination. As the wavelength of an
electron can be up to 100,000 times shorter than that of visible
light photons, electron microscopes have a higher resolving
power than light microscopes and can reveal the structure of smaller
objects. A transmission electron microscope can achieve better than
50 pm resolution and magnifications of up to about 10,000,000x whereas
most light microscopes are limited by diffraction to about
200 nm resolution and useful magnifications below 2000x.
• Electron microscopes are used to investigate the ultrastructure of a wide
range of biological and inorganic specimens including
microorganisms, cells, large molecules, biopsy samples, metals,
and crystals. Industrially, electron microscopes are often used for quality
control and failure analysis. Modern electron microscopes produce
electron micrographs using specialized digital cameras and frame
grabbers to capture the image.
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6. SCANNING ELECTRON
MICROSCOPE (SEM)
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7. PRINCIPLE OF SEM
• A scanning electron microscope (SEM) is a type
of electron microscope that produces images
of a sample by scanning it with a focused
beam of electrons. The electrons interact with
atoms in the sample, producing various
signals known as secondary electrons that
contain information about the sample's
surface topography and composition.
• SEM can achieve resolution better than 1
nanometre. Specimens can be observed in
high vacuum, in low vacuum, in wet conditions
(in environmental SEM), and at a wide range of
cryogenic or elevated temperatures.
• A wide range of magnifications is possible,
from about 10 times (about equivalent to that
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8. Components
of SEM
Emission
chamber
Electromagnetic
lenses
Specimen
chamber
• Electron gun :-
which emits
electron
• Condenser Lens
system – (focus
the electron
beam to a
narrower area).
• Objective
Aperture –
(directs the
narrowed beam
to objective
lens).
• Objective lens –
(captures the
electron beam
emerging form
the object).
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• The Detector –
(detects the
electrons
deflected by
the specimen).
• Television
Picture Tube –
(image is
formed on the
television
screen).

9. CONSTRUCTION OF SEM/INSTRUMENTATION
• Emission chamber - it is an electron generating arrangement used to generate
an electron –beam in TEM called as electron gun.This electron gun has a tungsten
filament which functions as cathode. It emits electron from its surface when it gets
heated by passage of high voltage electricity.
Tungsten is normally used in electron guns because it has the
highest melting point and lowest vapour pressure of all metals, and
because of its low cost. Other types of electron emitters
include lanthanum hexaboride (lab6) and zirconium oxide
cathodes.
• Electromagnetic lens systems – In SEM’s a radially symmetrically
coil of wire is used as the lens with current passing through it, such
virtual lenses are known as EM lenses.
• The coil is made up of few thousand turns of the wire having a soft
iron casting around it. An 1 amp. Current is passed through the coil
to produce EM field and the outer iron casting concentrates the
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10. TYPES OF EM LENS
i. Condenser lens – there are 2 condenser placed just
below the electron gun i.e. Primary condenser lens and
secondary condenser lens. It’s main function is to focus
the electron beam to a narrower part.
ii. Objective aperature – it directs the narrowed electron
beam which has the thickness of 20nm on to the
objective lens through the scanning coil.
iii. Scanning coil – The beam passes through pairs of
scanning coils or pairs of deflector plates in the electron
column, typically in the final lens, which deflect the
beam in the x and y axes so that it scans in
a raster fashion over a rectangular area of the sample
surface.
iv. Objective lens – it focuses the scanning beam on the
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11. SPECIMEN CHAMBER
• The detectors - the electrons are detected by
an everhart-thornley detector, which is a type
of scintillator-photomultiplier system. The
secondary electrons are collected by a positively
charged grid arrangement. In modern SEM’s
multi-electronic detectors are used to develop
coloured image instead of panchromatic
images.
• Television picture tube – the secondary electron
collected by thee positively charge grid is sent
to the television picture tube where the final
image is formed. The number of scanning lines
in SEM are far more as compared to an ordinary
television, this makes the picture very clear 17-01-2017
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12. WORKING OF SEM
• In a typical SEM, an electron beam is emitted from an electron gun fitted with
a tungsten filament cathode. Tungsten is normally used in electron guns
because it has the highest melting point and lowest vapour pressure of all
metals, and because of its low cost. Other types of electron emitters
include lanthanum hexaboride (lab6) and zirconium oxide cathodes. The
electron beam, which typically has an energy ranging from 0.2 kev to 40 kev, is
focused by one or two condenser lenses to a spot of sample. The beam passes
through pairs of scanning coils or pairs of deflector plates in the electron
column. In the final lens, the deflected beam in the x and y axis so that it can be
scanned in a raster fashion over a rectangular area of the sample surface.
• When the primary electron beam interacts with the sample, the electrons lose
energy by repeated random scattering and absorption within a teardrop-shaped
volume of the specimen known as the interaction volume, the interaction
volume depends on the electron's landing energy, the atomic number of the
specimen and the specimen's density. The energy exchange between the
electron beam and the sample results in the reflection of high-energy electrons
by elastic scattering, each of which can be detected by specialized detectors.
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13. APPLICATIONOFSEM
Biotechnology/
life sciences
• cell morphology,
• Development of
biocompatible
materials,
• tissue engineering
research,
• microbiology
Medical
sciences/
Research
• Determining the
receptors of cell.
• Identification of
Antigen-antibody
complex.
Semiconductor
industry
• Failure Analysis of
Integrated
Circuits
• Advanced
Packaging: Wire
Bonding
• Circuit Edits
• Micro-Electro-
Mechanical
Systems
Material
Sciences
• Steels & Metal
Alloys
• Polymers and
Composites
• Materials for
Building and Civil
Engineering
• Composition of -
Wood, Textile.
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Geology and
Earth
sciences.
• metamorphic
petrology and
mineralogy
• Ore processing
• Oil & Gas
• Palaeontolo-gy

14. LIMITATIONS OF SEM
• The disadvantages of a scanning electron microscope
start with the size (not portable) and cost (expensive).
• SEM’s are expensive, large and must be housed in an area
free of any possible electric, magnetic or vibration
interference.
• Maintenance involves keeping a steady voltage, currents
to electromagnetic coils and circulation of cool water.
• Special training is required to operate an SEM as well as
prepare samples.
• The preparation of samples can result in artifacts. The
negative impact can be minimized with knowledgeable
experience researchers being able to identify artifacts
from actual data as well as preparation skill. There is no
absolute way to eliminate or identify all potential
artifacts.
• In addition, SEM’s are limited to solid, inorganic samples
small enough to fit inside the vacuum chamber that can
handle moderate vacuum pressure.
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15. TRANSMISSION ELECTRON MICROSCOPE
(TEM)
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16. PRINCIPLE OF TEM
• Illumination source is a beam
of high velocity electrons
accelerated under vacuum,
focused by condenser lens
(electromagnetic bending of
electron beam) onto specimen.
• Image formation is due to loss
and scattering of electrons by
individual parts of the
specimen. Emergent electron
beam is focused by objective
lens. Final image forms on a
fluorescent screen for viewing.
• Image is captured on negative
or by digital camera
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17. Components
of TEM
Emission
chamber
Electromagnetic
lenses
Specimen
chamber
Photographic
arrangement
Vacuum
system
• Electron gun :-
which emits
electron • Condenser Lens
system – (focus
the electron
beam on the
object).
• Objective lens –
(captures the
electron beam
emerging form
the object).
• Additional Lens
– (Magnifies the
changes in the
object).
• Projector Lens –
(Collects the
magnified image
and projects it
on the florocent
screen)
• Air Lock system –
(Maintains the
vaccum in the
specimen
chamber).
• Microgrid –
(Holds the
specimen in its
place).
• Specimen Holder
– (Ensures the
alingment of
specimen in the
path of electron)
• Rotary Pump – (Creats
initial low vaccum).
• Oil Diffusion Pump –
(maintains the high
vaccum).
• Coldfingure – (Establishes
high vaccum).
• A plate
camera –
(enlarges the
image
produced by
the camera).
• 35 mm film
camera or
digital
camera
(captures the
image
projected on
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18. CONSTRUCTION OF TEM/ INSTRUMENTATION
1. EMISSION CHAMBER –
It is an electron generating arrangement used to generate
an electron –beam in TEM called as electron gun
This electron gun has a tungsten filament which
functions as cathode. It emits electron from its surface
when it gets heated by passage of high voltage electricity.
It is safely housed in a metallic chamber known as
cathode shield.
The anode consist of a metal plate with a hole drilled in
the centre. The circular hole of the anode is exactly in the
line with the circular hole of the cathode shield. Thus the
path of the electron beam is defined
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19. ELECTROMAGNETIC LENSES
• In TEM a radially symmetrically coil of wire is
used as the lens with current passing through it,
such virtual lenses are known as EM lenses.
• The coil is made up of few thousand turns of
the wire having a soft iron casting around it. An
1 amp. Current is passed through the coil to
produce EM field and the outer iron casting
concentrates the magnetic field.
• The focal length of the lens can be adjusted by
adjusting the current passing through it.
• There are various types of EM lenses systems
are used in tem like condenser lens system,
objective lens, additional lens, projector lens
etc.
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20. TYPES OF EM LENS SYSTEMS
i. Condenser lens system – it is located just below the
electron gun assembly. And its main function is to
condense (focus) the electron beam on the object. In
earlier versions there were only one condenser lens
placed but in Morden TEM’s there are 2 condenser
lenses.
ii. Objective lens – it is places just below the specimen
chamber and its main function is to capture the electron
beam emerging from the objects.
iii. Additional lenses – these are also called as intermediate
lenses which are placed below the objective lens, these
lenses allows the magnification of the diffraction pattern
of the electrons caused by the structure of the
specimen.
iv. Projector lens – it resembles to that of a eye piece of a
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21. SPECIMEN CHAMBER
• The specimen is to be place exactly in the centre
between the condenser and objective lens.
• In an electron microscope the changing of specimen
should be carried out without breaking the vacuum
present in the column of the microscope, for this the
specimen chamber is provided with an air-lock system.
• The sections used in tem must be ultra thin having the
thickness less than 1 µ. These thin sections are to be
mounted on a microgrid, which is placed on the
specimen holder.
• The specimen holder is a metallic block which assures
that the specimen mounted on the microgrid is in the
electron path. 17-01-2017ELECTRON MICROSCOPY - SEM & TEM 21

22. VACUUM SYSTEM
• Vacuum is developed at two levels – a standard rotary pump is
used to develop initial low vacuum and for high vacuum a
metal piece is inserted in the vacuum system to produce high
vacuum and is known as cold-fingure.
• This metal piece before inserting is treated with liquid nitrogen
which traps the air molecules and gasses on its cool surface.
• Oil diffusion pumps are used to maintain this high vacuum.
This vacuum generated provides an air-lock column where the
specimen is mounted.
PHOTOGRAPHIC
ARRANGEMENT
• It consist of a plate camera which is situated
below the florescent screen as it enlarges
the photographed image on the screen.
• An additional 35mm film camera or a digital
camera is fixed to capture the image
projected on the screen.
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23. WORKING OF TEM
• The original form of electron microscope, the transmission electron
microscope (TEM) uses a high voltage electron beam to illuminate the specimen
and create an image. The electron beam is produced by an electron gun,
commonly fitted with a tungsten filament cathode as the electron source. The
electron beam is accelerated by an anode typically at +100 ev (40 to 400 kev) with
respect to the cathode, focused by electrostatic and electromagnetic lenses, and
transmitted through the specimen that is in part transparent to electrons and in
part scatters them out of the beam. When it emerges from the specimen, the
electron beam carries information about the structure of the specimen that is
magnified by the objective lens system of the microscope. The spatial variation in
this information (the "image") may be viewed by projecting the magnified
electron image onto a fluorescent viewing screen coated with
a phosphor or scintillator material such as zinc sulfide. Alternatively, the image
can be photographically recorded by exposing a photographic
film or plate directly to the electron beam, or a high-resolution phosphor may be
coupled by means of a lens optical system or a fibre optic light-guide to the sensor
of a digital camera. The image detected by the digital camera may be displayed on
a monitor or computer. 17-01-2017ELECTRON MICROSCOPY - SEM & TEM 23

24. CHECKLIST FOR TEM
 Various lenses and the electron gun should be accurately
lined up on a common axis. The specimen should be less
than 1µ in size.
 After placing the specimen in the specimen chamber fine
micrometre screws are to be used to move the specimen in
the line of electron beam.
 The focal length of the EM lenses varies with the
wavelength of electron and intensity of the electric current
flowing through so the electric flow should be stabilized
using a stabilizer.
 The electric current can also be maintained by creating
resistance for the flow.
 Morden TEM’s have aberration correctors which reduces
the distortion in the image. They also have a
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25. APPLICATIONSOFTEM Material Sciences
• morphology, structure, and local chemistry of metals
• investigation of crystal structures, orientations and chemical
compositions of phases, precipitates and contaminants
Geology, Environmental Science
• To study the mineral composition.
• To study the toxic effect at molecular level.
• Study of composition of Mineral from ore
Medical and life Sciences
• To study the structure and composition of viruses (virology)
• To study the composition of cell. 17-01-2017ELECTRON MICROSCOPY - SEM & TEM 25

26. LIMITATIONS
• Many materials require
extensive sample preparation
to produce a sample of 1µ
which is time consuming.
• The sample preparation may
bring structural changes in the
original structure.
• As the field of view is very
small so the area or the region
of the sample observed may
not represent the whole.
• Biological samples may get
damaged on prolonged
exposure to electron beam.
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27. WHICH ONE IS BETTER ?
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28. 17-01-2017ELECTRON MICROSCOPY - SEM & TEM 28
SEM’s
• SEM images are artificially coloured these days
by false colour contrast (FCC) process. This may
be done for aesthetic effect, to clarify structure
or to add a realistic appearance to the
sample and generally does not add information
about the specimen.
• The third dimension images can be
reconstructed by collaging some series of stereo
pairs captured from with different angles.
• The environmental scanning electron
microscope is a scanning electron
microscope (SEM) that allows for the option of
collecting electron micrographs of specimens
that are "wet," uncoated, or both by allowing for
a gaseous environment in the specimen
chamber.
TEM’s
• TEM’s are combined with SEM’s band dark field
imaging has been reported in the generally low
accelerating beam voltage range used in SEM,
which increases the contrast of unstained
biological specimens at high magnifications with
a field emission electron gun.
• Earlier version of TEM’s had only one condenser
lens but in Morden TEM’s there are 2 condenser
lens followed aberration correctors which
reduces the distortion in the image.
• They also have a monochromater to reduce the
energy spread of the incident electron beam less
than 0.15ev.
Advancements in TEM & Sem

29. REFERENCES
• HTTPS://EN.WIKIPEDIA.ORG/WIKI/ENVIRONMENTAL_SCANNING_ELECTRON_MICR
OSCOPE
• HTTPS://EN.WIKIPEDIA.ORG/WIKI/SCANNING_ELECTRON_MICROSCOPE
• HTTP://WWW.MICROSCOPEMASTER.COM/TRANSMISSION-ELECTRON-
MICROSCOPE.HTML
• HTTP://WWW.SEM-APPLICATIONS.COM/
• HTTPS://WWW.GOOGLE.CO.IN/SEARCH?Q=SEM&ESPV=2&BIW=1242&BIH=557&
TBM=ISCH&SOURCE=LNMS&SA=X&VED=0AHUKEWIIU6TCIMFRAHUIQ48KHZJOAJ
AQ_AUIBIGB
• HTTPS://WWW.GOOGLE.CO.IN/SEARCH?Q=SEM&ESPV=2&BIW=1242&BIH=557&
TBM=ISCH&SOURCE=LNMS&SA=X&VED=0AHUKEWIIU6TCIMFRAHUIQ48KHZJOAJ
AQ_AUIBIGB#TBM=ISCH&Q=TEM
• HTTPS://WWW.GOOGLE.CO.IN/SEARCH?Q=SEM&ESPV=2&BIW=1242&BIH=557&
TBM=ISCH&SOURCE=LNMS&SA=X&VED=0AHUKEWIIU6TCIMFRAHUIQ48KHZJOAJ
AQ_AUIBIGB#TBM=ISCH&Q=IMAGE+IN+SEM+AND+TEM 17-01-2017
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