The document provides an overview of microscopy, detailing the history and types of electron microscopes, specifically the Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM). It discusses their components, working principles, advantages, limitations, and applications in various scientific fields. Additionally, it highlights the differences between light microscopy and electron microscopy, emphasizing the advancements brought by electron microscopy in resolving power and magnification capabilities.

1. PRESENTED BY:
Subhankar Das
3rd Semester
B.Sc Biotechnology

2. 1- Introduction to Microscopy
2- Electron Microscope
History of Electron Microscope
Types of electron Microscope
3- Transmission Electron Microscope
History of TEM
Components of TEM
Sample Preparation in TEM
Working of TEM
Advantages of TEM
Limitation of TEM
Applications of TEM
4- Scanning Electron Microscope
History of SEM
Components of SEM
Sample Preparation in SEM
Working of SEM
Advantages of SEM
Limitation of SEM
Applications of SEM
4- Difference between Light and Electron
Microscope.
5- Difference between TEM & SEM.

3. An optical instrument used for viewing very small objects, such as
mineral samples or animal or plant cells, typically magnified several
hundred times.
The branch of science that deals with the study of the construction,
working principles and use of microscopes.

4. An electron microscope is a type of microscope that uses an
electron beam to illuminate a specimen and produce a
magnified image.
Advantages of electron microscope over light microscope :
 An EM has greater resolving power than ordinary one.
 Can reveal the structure of smaller objects because electrons have
wavelengths about 100,000 times shorter than visible light .
 They can achieve magnification of up to about 10,000,000x whereas
ordinary, light microscopes are about 200 nm resolution and useful
magnifications below 2000x.

5. HISTORY.
Hertz (1857-94) suggested that cathode rays were a form of wave motion .
It had earlier been recognized by Plücker in 1858 that the deflection of "cathode
rays" (electrons) was possible by the use of magnetic fields. This effect had been
utilized to build primitive cathode ray oscilloscopes (CROs) as early as 1897 by
Ferdinand Braun, intended as a measurement device.
Weichert, in 1899, found that these rays could be concentrated into a small spot by
the use of an axial magnetic field produced by a long solenoid.
In 1926 that Busch showed theoretically that a short solenoid converges a beam of
electrons in the same way that glass can converge the light of the sun.
Busch should probably therefore be known as the father of electron optics.

6. 1. Transmission electron microscope (TEM)
2. Scanning electron microscope (SEM)
3. Reflection electron microscope (REM)
4. Scanning transmission electron microscope (STEM)

8. Ernst Ruska developed the first
electron microscope, a TEM,
with the assistance of Max
Knolls in 1931.
After significant improvements
to the quality of magnification,
Ruska joined the Sieman’s
Company in the late 1930s as
an electrical engineer, where he
assisted in the manufacturing
of his TEM
The first commercial TEM
was available in 1939.

9. Electron gun
Vacuum system
Electromagnetic lens
Camera and display
Specimen stage

10. From the top down, the TEM
consists of an emission source,
which may be a
, or a
source.
For tungsten, this will be of
the form of either a hairpin-
style filament, or a small spike-
shaped filament and for LaB6
sources utilize small single
crystals.
By connecting this gun to a
high voltage source (typically
~100–300 kV) the gun will,
begin to emit electrons.

11. are the first in the
series. They are also called the
"roughing pumps" as they are used to
initially lower the pressure within the
column through which the electron
must travel to 10 -3 mm of Hg range.
may achieve
higher vacuums (in the 10-5 mm Hg
range) but must be backed by the
rotary pump.
In addition a
, when an even greater
vacuum is required.

12. Electromagnetic lenses are made of a
coil of copper wires inside several iron
pole pieces .
Magnetic condenser lens: -
converged the electron beam on the
specimen.
Magnetic objective lens: - Focuses
the electron into the first real image of
the object which is enlarged 2000
times.
Magnetic intermediate lens : -forms
an intermediate image of the specimen
Magnetic projector lens: - it then
magnifies a portion of the first image

13. An image is formed from the
interaction of the electrons
transmitted through the
specimen; the image is
magnified and focused onto an
imaging device, such as:-
A fluorescent screen
On a layer of photographic
film, or to be detected by a
sensor such as a CCD
"Charged Coupled Device"
camera.

14. The specimen holders are adapted to hold a
standard size of grid upon which the sample is
placed or a standard size of self-supporting
specimen.
Usual grid materials are copper,
molybdenum, gold or platinum. This grid is
placed into the sample holder, which is paired
with the specimen stage.
The most common is the side entry holder,
where the specimen is placed near the tip of a
long metal (brass or stainless steel) rod, with
the specimen placed flat in a small bore.
Along the rod are several polymer vacuum
rings to allow for the formation of a vacuum
seal of sufficient quality, when inserted into the
stage.

15. 1. Fixation: The first step in sample preparation, has the aim
of preserving tissue in its original state. Fixatives must
be buffered to match the pH and osmolarity of the living
tissue. Eg . Glutaraldehyde
2. Dehydration: - Before sample can be transferred to resin all
the water must be removed from the sample. This is carried
out using a graded ethanol series.
3. Tissue sectioning: -By passing samples over a glass or
diamond edge, small, thin sections can be readily obtained
using a semi-automated method.
Contd…

16. • Sample staining: - Details in light microscope
samples can be enhanced by stains that absorb light;
similarly TEM samples of biological tissues can utilize
high atomic number stains to enhance contrast. The
stain absorbs electrons or scatters part of the
electron beam Compounds of heavy metals such as
osmium, lead, uranium or gold (in immunogold
labelling) may be used
• Mechanical milling: -Mechanical polishing may be
used to prepare samples. Polishing needs to be done
to a high quality, to ensure constant sample
thickness across the region of interest. A diamond, or
cubic boron nitride polishing compound may be used

17. The electron source is commonly a tungsten filament
of 30-150 KV potential. The electron beam passes
through the centre of ring-like magnetic condenser
and becomes converged on the specimen. After being
transmitted through the specimen, the magnetic
objective focuses the electron into the first (real)
image of the object which is enlarged (2000 times).
The magnetic projector lens then magnifies a portion
of the first image producing magnification up to 240,
000 or more.
The final enlarged image can be viewed by
striking on fluorescent screen which makes it visible.
The image can also be thrown upon a photographic
plate for permanent record.
Molecules in the microscope interfere with
the movement of electrons. To prevent this, the
interior of the microscope is kept in the state of high
vacuum, around 10.4-10.6 mmHg. It is also necessary
to have specimen ultra thin. The electron beams have
a poor penetrating power, therefore, only small
objects or very thin sections of the specimen can be
examined.

19. • TEMs offer the most powerful magnification, potentially over
one million times or more.
• TEMs have a wide-range of applications and can be utilized in a
variety of different scientific, educational and industrial fields.
• TEMs provide information on element and compound structure.
• Images are high-quality and detailed.
• TEMs are able to yield information of surface features, shape,
size and structure.
• They are easy to operate with proper training.

20. • TEMs are large and very expensive.
• Laborious sample preparation.
• Potential artifacts from sample preparation.
• Operation and analysis requires special training.
• Samples are limited to those that are electron
transparent, able to tolerate the vacuum chamber and
small enough to fit in the chamber.
• TEMs require special housing and maintenance.
• Images are black and white.

21. 1. Cancer research
2. Virology
3. Materials science as well as pollution
4. Nanotechnology and
5. Semiconductor research.

23. The earliest known work
describing the concept of a
Scanning Electron Microscope
was by M. Knoll (1935).
In 1965 the first commercial
instrumented SEM by
Cambridge Scientific Instrument
Company as the "Stereoscan”.
The first SEM used to examine
the surface of a solid specimen
was described by Zworykin et al.
(1942).

24. Electron Gun
Electromagnetic lens
Scan coil
Vacuum system
Detectors

25. Tungsten is normally used in
thermionic electron guns because it has
the highest melting point and lowest
vapour pressure of all metals, thereby
allowing it to be heated for electron
emission, and because of its low cost.
Other types of electron emitters
include lanthanum hexaboride (LaB
6) cathodes.
In a typical SEM, an electron beam is
thermionically emitted from an electron
gun fitted with a tungsten filament
cathode.

26. There are two main lenses used
in SEM: -
1. Condenser lenses
2. Objective lenses
Main role of the condenser lens
is to control the size of the beam
The objective lens focuses
electrons on the sample at the
working distance

27. A high vacuum minimises
scattering of the electron beam
before reaching the specimen.
This is important as scattering or
attenuation of the electron beam will
increase the probe size and reduce
resolution, especially in the SE
mode.
The high vacuum condition also
optimises collection efficiancy,
especially of the secondary
electrons.

28. An electron detector is
placed in the sample
chamber.
By having a 10 keV
positive potential on its face,
it attracts the secondary
electrons emitted from the
sample surface.
One advantage of this
biased detector is that it can
attract secondary electrons
emitted from sides of the
sample which are physically
blocked from the detector
face.
This greatly reduces
shadowing effects in SEM
images.

29. • Some samples, such as hard tissues like bone or
teeth, and organisms with a tough exoskeleton, such
as some arthropods, can be studied without any
preparation, but these are the exception.
• Biological specimens, such as cells and tissues or
tissue components, must first be fixed to preserve
their native structure.
• Fixation is done either by chemical or physical
means.
contd. . .

30. • Chemical fixation uses formalin or glutaraldehyde of
varying per cent concentrations in a buffer of a specific
pH and osmolarity. Physical fixation may be by heat (such
as boiling an egg), but is more commonly done by
freezing.
• Hydrated samples, like most biological and some
materials specimens, must first be dehydrated before
placing the specimen in the SEM sample chamber. This is
typically done by passing the specimens through a
graded series of ethanol-water mixtures to 100%.
• If the specimen is an electron conductor, it needs only to
be held on an appropriate support. If it is non-conductor
it is allowed to dry but if moist, freeze dried in liquid
nitrogen is necessary. The specimen is then coated with
metal vapour (gold) in vacuum.

31. The scanning electron microscope gives 3D
(dimensional surface) views of objects.
The electron originates at high energy
(20,000 v) from a hot tungsten cathode
“gun”. These electrons are sharply focused,
adjusted and narrow by an arrangement of
magnetic fields. The primary beam (Probe)
acts only as an exciter of image forming
secondary electrons emerging from the
surface of the specimen. The probe scans
the specimen. Images are elicited from
wherever the probe strikes the metal coated
areas of the specimen.
The useful secondary electrons are
magnetically deflected to a collector or
detector. The successive signal from the
collector are amplified and transmitted to a
cathode ray (T.V.) tube. The scanning beam
and T.V. tube beam are synchronized. The
image scans by the eye on T.V. screen. The
T.V. image may be photographed,
videotaped or processed in motion on a
computer.

32. 1. Its wide-array of applications.
2. The detailed three-dimensional and topographical imaging
and the versatile information gathered from different
detectors.
3. This instrument works fast.
4. Although all samples must be prepared before placed in
the vacuum chamber, most SEM samples require minimal
preparation actions.

33. • The disadvantages of a Scanning Electron Microscope start with the size and
cost.
• SEMs 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.

34. 1. Crystallography
2. Chemistry
3. Microstructure studies
4. Surface contamination examination
5. IC failure analysis

40. Electron Microscopy and Analysis by : P.J. GOODHEW, University
of surrey and F.J.Humphreys, UK imperial College, London, UK
Wikipedia, en.wikipeddia.org
Hawkes, P. ,The beginnings of Electron microscopy
Transmission Electron Microscopy and Diffractometry of Materials.
Springer. 2007. ISBN 3540738851
Hubbard, A Electron Diffraction in the Transmisssion Electron
Microscope. Garland Science. ISBN 1859961479
Tanaka, Nobuo “ Present status and future prospects of spherical
aberration corrected TEM/STEM for study of nanomaterials” (free
download review).
Adrian, Marc; Dubochet, Jacques; Lepault, Jean; McDowall,
Alasdair W “Cryo-electron microscvopy of viruses”.