The document provides an overview of scanning electron microscopes (SEMs), including their history, key parts, working principle, applications, and sample preparation process. Some key points: - SEMs use a beam of electrons to produce high-resolution images of sample surfaces, allowing examination of microscopic structural features. They have greater depth of field than light microscopes. - Early development began in the 1930s. Commercial instruments became available in the 1960s. Continued improvements have increased resolution to the atomic scale. - Key components include an electron gun, electromagnetic lenses, vacuum system, specimen stage, and detectors. Secondary electrons emitted from the sample are used to form images. - Applications span biology, materials

1. GOOD MORNING !
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2. Scanning Electron
Microscope
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3. CONTENTS:
1. Introduction
2. History
3. Applications
4. Principle
5. Parts
6. Preparation of sample
7. Advantages and disadvantages
8. References
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4. Introduction :
The scanning electron microscope is one of the most
versatile instruments available for examination and analysis
of the microstructural characteristics of solid objects.
The primary reason for the SEMs usefulness is the high
resolution which can be obtained when bulk objects are
examined.
Another important feature of SEM images is the three
dimensional appearance of specimen, which is a result of
large depth of focus. 4

5. ➔ SEM can provide useful information about the
composition at the specimen surface.
➔ The user can obtain high magnification images, with
a good depth of field, and can also analyse individual
crystals or other features.
➔ A high-resolution SEM image can show detail down
to 25 Angstroms, or better.
➔ When used in conjunction with the closely-related
technique of energy-dispersive X-ray microanalysis
(EDX, EDS, EDAX), the composition of individual
crystals or features can be determined.
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7. ➔ The area to be examined is irradiated with a finely
focused beam of electrons which may be static or
swept in a raster across the surface of the specimen.
➔ They are obtained from specific emission volumes
within the sample and are used to measure many
characteristics of the sample (composition, surface
topography , crystallography,)
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8. The resultant images are 3 dimensional and have better
depth of field.
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9. History of microscope
Zacharias Jansen Anton van
Leeuwenhoek
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10. The first operational electron microscope
was presented by Ernst Ruska and Max
Knoll in 1932, and 6 years later Ruska had a
first version on the market. In 1986 Ruska
received a Nobel Prize in physics for his
"fundamental work in electron optics and
for the design of the first electron
microscope".
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11. The invention of the electron microscope by Max
Knoll and Ernst Ruska at the Berlin Technische
Hochschule in 1931 finally overcame the barrier to
higher resolution that had been imposed by the
limitations of visible light. Since then resolution
has defined the progress of the technology. The
ultimate goal was atomic resolution - the ability to
see atoms. To avoid the effects of objective lens
chromatic aberration with thick samples in TEM
, SEM was invented.
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12. It was Manfred von Ardenne in 1937 invented
SEM. Further work was reported by Zworykin's
group, followed by the Cambridge groups in the
1950s and early 1960s headed by Charles Oatley,
all of which finally led to the marketing of the
first commercial instrument by Cambridge
Scientific Instrument Company as the
"Stereoscan" in 1965, which was delivered to
DuPont.
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13. Applications of SEM:
Scanning Electron Microscopy - SEM - is a
powerful technique in the examination of
materials. It is used widely in metallurgy,
geology, biology and medicine, to name just a few.
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14. APPLICATIONS
● Biomedical engineering
● Cell & Tissue morphology
● Microbiology
● Pharmaceuticals
● Plant and animal biology
● Subcellular analysis
● Environmental and Food Sciences
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15. BIOMEDICAL ENGINEERING
SEMs combine the latest concepts from medicine,
biotechnology and engineering for designing a
variety of technologies such as support matrices for
cell growth, artificial tissue and implantable
biomedical device.
Growth of osteoblasts on
zirconia ceramics
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16. CELL & TISSUE MORPHOLOGY
SEMs are the ideal instruments for investigating cellular and
tissue structure with high resolution. Typical applications involve
observing shape changes of grooves, pores, blebs or microvilli on
the cellular in response to the changes in the extracellular
environment.
Detail of a fibroblast cell
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17. MICROBIOLOGY
High resolution imaging of the microbial surface using SEM
helps to have a better understanding of the morphology of
microbial populations, bacteria communication and biofilm
formation. SEMs help researchers visualize microbial
populations with great focal depth and high resolution.
S.mutans on dental
filling
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18. SUBCELLULAR ANALYSIS
The SEM technology is becoming more popular in this field, due to
emerging techniques which are available to scanning electron
microscopy. SEM offers several solutions for scientists interested in
the subcellular investigations of biological samples.
Osteoblast layer in a
mouse tooth.
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19. Principle
In a scanning electron microscope, the specimen
is exposed to a narrow electron beam from an electron
gun, which rapidly moves over or scans the surface of the
specimen.
This causes the release of a shower of secondary electrons
and other types of radiations from the specimen surface.
The intensity of these secondary electrons depends upon
the shape and the chemical composition of the irradiated
object. These electrons are collected by a detector, which
generates electronic signals.
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22. Construction of SEM :
1. Electron operating system
2. Specimen stage
3. Secondary electron detector
4. Image display unit
5. Operation system
Electron gun
Condenser lens
Objective lens
Scanning coil
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24. Electron Gun :
➢ Electron gun produces an electron beam.
➢ Thermo electrons are emitted from a filament(cathode) made
of thin tungsten wire by heating the filament to a high
temperature.
➢ These thermo electrons are gathered as an electron beam
flowing into the metal plate(anode) by applying positive
voltage to anode.
➢ If a hole is made at the centre of the anode, the electron beam
flows through it.
➢ An electrode is placed between cathode and anode to adjust
the current of electron beam.
➢ The electron beam is finely focused by the action of wehnelt
electrode.
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26. Construction of lens :
➢ A direct electric current is passed through
a coil wound electric wire a rotationally
symmetric magnetic field is formed and a lens
action is produced on electron beam.
➢ The surrounding of the coil is enclosed by
yoke so that part of magnetic field leaks out
from a narrow gap.
➢ A portion with narrow gap is called pole
piece.
➢ When the current passing through the coil changes, the strength
of coil is also changed which is not possible in light microscope.
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27. Lenses
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28. Condenser lens and objective lens
● Two stage lenses which combine the condenser and objective
lenses are located below the electron gun.
● The electron beam from the electron gun is focused by the 2 stage
lenses and a small electron probe is produced.
● Placing a lens below the electron gun enables you to adjust the
diameter of electron beam.
● A fine electron beam is required for SEM.
● The aperture is placed between condenser and objective lens.
● The aperture is made of thin metal plate and has a small hole.
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30. ● The electron beam, which passed through the
condenser lens illuminates this aperture plate.
● The aperture allows a part of electron beam to
reach the objective lens.
● The objective lens is used for focusing.
● It determines the final diameter of electron
probe.
● If the objective lens is not good, an optimally
fine electron probe cannot be produced .
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31. Specimen stage
➔ In general, the specimen is observed at higher
magnification in an electron microscope.
➔ Thus a specimen stage which stably supports the
specimen and moves smoothly is required.
➔ The specimen stage in SEM can perform following
movements:
- Horizontal movement
- Vertical movement
- Specimen tilting
- Rotation
Change of image resolution
Selection of field of view
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33. ● Most SEMs use eucentric specimen
stage.
● By the use of this stage the specimen
does not change after shifting the
field of view when the specimen is
tilted.
● In addition to manual drive stage, the
use of motor driven stage has
increased.
● In computer controlled specimen
stage, the stage can be moved to
selected point by simply clicking
mouse and restore the stage to
desired observed point.
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34. Secondary electron detector
➔ The secondary electron detector detects secondary electrons emitted
from specimen invented by Everhart and Thornley. Also called E-T
detector.
➔ A scintillator (fluorescent) substance is coated on the tip of the
detector and a high voltage of 10kv is applied to it.
➔ The secondary electrons from the specimen are attracted to this high
voltage and then generate light when they hit the scintillator.
➔ This light is directed to a photomultiplier tube (PMT) through a light
guide.
➔ Then the light is converted to electrons and these electrons are
amplified as an electric signal.
➔ This is present in sample chamber nearer to objective lens.
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39. Preparation of sample
➔ Treatment of biological specimens involves
following procedure:
- Removing and cleaning of tissues
- Fixation
- Dehydration
- Drying
- Mounting and coating
Coating is same as for other non conductive
specimens. It is coated with a thin metal film so that
surface has conductivity.
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Coating
➔ The dried specimen is mounted on an aluminium stub.
➔ A simple adhesive like durofix may be used.
➔ The specimen is placed in the chamber of a sputter
coating unit and air is evacuated by purging the
chamber with argon for 2 mins.
➔ When vacuum of 0.1torr is attained gold is discharged
from a target situated above the specimen and
deposited on the surface of sample.
➔ The thickness of the layer of gold is determined by the
distance of the specimen from the target, period of time
taken to sputter and strength of current applied to
target.

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Cryopreservation:
➔ Eliminates the artefacts associated with fixation,
dehydration and embedding.
➔ There are now cryosystems which interface directly to
SEM which allow fresh tissue to be frozen rapidly with
liquid nitrogen, sputtered with gold and then examined in
SEM while it is still frozen.
➔ These cryosystems consists of
- Slushing chamber to freeze specimen
- Low temperature preparation chamber in which
specimens may be fractured have the ice sublimed from
their surface and be coated. This preparation chamber
can be fitted directly to SEM or may be a separate unit.

42. Differences between TEM and SEM
SEM TEM
Scattered electrons Transmitted electrons
Larger samples can be examined. Sample has to be cut into thinner
sections.
Large amount of sample can be
examined at a time.
Only small amount of sample can
be examined at a time.
Comparatively lesser resolution. Resolution is greater than SEM.
Effective Instrument Resolution -
1nm
Effective Instrument Resolution -
0.5nm 42

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46. Journals
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References :
● Cullings handbook of histopathological and histochemical techniques - 3rd
edition.
● Bancroft book of histopathology - 7th edition.
● Various internet sources

54. Thanks!
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