• This is a powerful electron microscope that uses a beam of

electrons to focus on a specimen producing a highly magnified

and detailed image of the specimen.
• The magnification power is over 2 million times better than
that of the light microscope, producing the image of the
specimen which enables easy characterization of the image in
its morphological features, compositions and crystallization
Introduction

information is also detailed.

• This TEM microscope has several advantages compared to the
light microscope with its efficiency also being very high.
• Among all microscopes both light and electron microscopes,
TEM are the most powerful microscopes used in laboratories. It
can magnify a mall particle of about 2nm, and therefore they
have a resolution limit of 0.2um.
• The working principle of the Transmission Electron
Microscope (TEM) is similar to the light microscope.
The major difference is that light microscopes use
light rays to focus and produce an image while the Principle of Transmission
TEM uses a beam of electrons to focus on the
specimen, to produce an image. Electron Microscope (TEM)
• Electrons have a shorter wavelength in comparison
to light which has a long wavelength.
• The mechanism of a light microscope is that an
increase in resolution power decreases the
wavelength of the light, but in the TEM, when the
electron illuminates the specimen, the resolution
power increases increasing the wavelength of the
electron transmission.
• The wavelength of the electrons is about 0.005nm
which is 100,000X shorter than that of light, hence
TEM has better resolution than that of the light
microscope, of about 1000times.
• This can accurately be stated that the TEM can be
used to detail the internal structures of the smallest
particles like a virion particle.
Parts of Transmission
Electron Microscope (TEM)

Their working mechanism is enabled by

the high-resolution power they produce
which allows it to be used in a wide
variety of fields. It has three working parts
which include:
1. Electron gun
2. Image producing system
3. Image recording system
• This is the part of the Transmission Electron Microscope
responsible for producing electron beams.
• Electrons are produced by a cathode that is a tungsten
Electron gun
filament that is V-shaped and it is normally heated. The
tungsten filament is covered by a control grid known as a
Wehnelt cylinder made up of a central hole which lies
columnar to the tube.
• The cathode lies on top of or below the cylindrical column
hole. The cathode and the control grid are negatively
charged with an end of the anode which is disk-shaped
that also has an axial hole.
• When electrons are transmitted from the cathode, they
pass through the columnar aperture (hole) to the anode at
high voltage with constant energy, which is efficient for
focusing the specimen to produce an accurately defined
image.
• It also has the condenser lens system which works to
focus the electron beam on the specimen by controlling
the energy intensity and the column hole of the electron
gun. The TEM uses two condenser lenses to converge the
beam of electrons to the specimen.
• The two condenser lens each function to produce an
image i.e the first lens which has strong magnification,
produces a smaller image of the specimen, to the second
condenser lens, directing the image to the objectives.
• Its made up of the objective lens, a
movable stage or holding the specimen, Image- Producing system
intermediate and projector lenses. They
function by focusing the passing electrons
through the specimen forming a highly
magnified image.
• The objective has a short focal length of
about 1-5mm and it produces an
intermediate image from the condenser
which are transmitted to the projector
lenses for magnification.
• The projector lenses are of two types, i.e
the intermediate lens which allows great
magnification of the image and the
projector lens which gives a generally
greater magnification over the intermediate
lens.
• To produce efficient high standard images,
the objectives and the projector lenses
need high power supplies with high stability
for the highest standard of resolution.
• Its made up of the fluorescent screen used to view and to Image-Recording System
focus on the image. They also have a digital camera that
permanently records the images captured after viewing.
• They have a vacuum system that prevents the
bombardment or collision of electrons with air molecules
disrupting their movement and ability to focus. A
vacuumed system facilitates the straight movement of
electrons to the image.
• The vacuumed system is made up of a pump, gauge,
valves and a power supply.
• The image that is formed is called a monochromatic
image, which is greyish or black and white. The image
must be visible to the human eye, and therefore, the
electrons are allowed to pass through a fluorescent
screen fixed at the base of the microscope.
• The image can also be captured digitally and displayed on
a computer and stored in a JPEG or TIFF format. During
the storage, the image can be manipulated from its
monochromatic state to a colored image depending on
the recording apparatus eg use of pixel cameras can store
the image in color.
• The presence of colored images allows easy visualization,
identification, and characterization of the images.
 How does a Transmission Electron Microscope (TEM) work?
From the instrumentation described, the working The optical electron beam diagram of TEM.
mechanism is a sequential process of the parts of the
TEM mentioned above. To mean:
•A heated tungsten filament in the electron gun
produces electrons that get focus on the specimen by
the condenser lenses.
•Magnetic lenses are used to focus the beam of electrons
of the specimen. By the assistance offered by the column
tube of the condenser lens into the vacuum creating a
clear image, the vacuum allows electrons to produce a
clear image without collision with any air molecules
which may deflect them.
• On reaching the specimen, the specimen scatters the electrons focusing them on the magnetic lenses forming
a large clear image, and if it passes through a fluorescent screen it forms a polychromatic image.
• The denser the specimen, the more the electrons are scattered forming a darker image because fewer
electron reaches the screen for visualization while thinner, more transparent specimens appear brighter.
• NOTE: If the screen is moved aside, a photographic image can be captured in pixels forming a permanent
image.
Preparation of specimen for visualization by TEM
• The specimens under study have to be thin if any information is to be obtained using
transmitted electrons in the TEM.
• For a sample to be transparent to electrons, the sample must be thin enough to transmit
sufficient electrons such that enough intensity falls on the screen to give an image.
• This is a function of the electron energy and the average atomic number of the
elements in the sample.
• Typically for 100 keV electrons, a specimen of aluminum alloy up to ~ 1 μm would be
thin, while steel would be thin up to about several hundred nanometers. However,
thinner is better and specimens < 100 nm should be used wherever possible.
• The method to prepare the specimens for TEM depends on what information is
required. In order to observe TEM images with high resolution, it is necessary to
prepare thin films without introducing contamination or defects.
• For this purpose, it is important to select an appropriate specimen preparation method
for each material, and to find an optimum condition for each method.
Crushing
• A specimen can be crushed with an agate mortar and pestle.
• The flakes obtained are suspended in an organic solvent (e.g., acetone),and
dispersed with a sonic bath or simply by stirring with a glass stick.
• Finally, the solvent containing the specimen flakes is dropped onto a grid. This
method is limited to materials which tend to cleave (e.g., mica).
Physical Aspects
• Describing the resolution of a TEM in terms of the classic Rayleigh criterion for VLMs, which states
that the smallest distance that can be investigated, δ, is given approximately by , where λ is the
wavelength of the electrons, μ is the refractive index of the viewing medium, and β is the semi-
angle of collection of the magnifying lens.

• According to de Broglie’s ideas of the wave-particle duality, the particle momentum p is related to its
wavelength λ through Planck’s constant h,

Momentum is given to the electron by accelerating it through a potential drop, V, giving it a kinetic energy,
eV. This potential energy is equal to the kinetic energy of the electron
• Based upon the foregoing, we can equate the momentum (p) to the electron mass (mo),
multiplied by the velocity (v) and substituting for v from above equations

• These equations define the relationship between the electron wavelength, λ, and the accelerating
voltage of the electron microscope (V), Eq. However, we have to consider about the relative effects
when the energy of electron more than 100 keV. So to be exact we must modify

• if a higher resolution is desired a decrease in the electron wavelength is accomplished by increasing

the accelerating voltage of the electron microscope. In other words, the higher accelerating rating
used, the better resolution obtained
 Why the Specimen Should be Thin
• All scattering in the TEM specimen is often
approximated as a single scattering event since it is the
simplest process. If the
• specimen is very thin, this assumption will be
reasonable enough. If the electron is scattered more
than once, it is called ‘plural
• scattering.’ It is generally safe to assume single
scattering occurs, unless the specimen is particularly
thick. When the times of
• scattering increase, it is difficult to predict what will
happen to the electron and to interpret the images and
DPs. So, the principle is
• ‘thinner is better’, i.e., if we make thin enough
specimens so that the single-scattering assumption is
plausible, and the TEM
• research will be much easier. Two different kinds of electron scattering form (a) a thin
specimen and (b) a bulk specimen.
 Electron Beam Damage

TEM images at the slit edge of the GaAs samples prepared by slit focused ion beam. GaAs samples
prepared at (a) 3 kV, (b) 5 kV, (c) 10 kV, (d) 20 kV, and (e) 30 kV. The thickness of the amorphous layer
produced by focused ion beam is shown in each image.
A detrimental effect of ionizing radiation is that it can damage the specimen, particularly polymers (and most
organics) or certain

minerals and ceramics. Some aspects of beam damage made worse at higher voltages. Figure shows an area of a
specimen

damaged by high-energy electrons. However, the combination of more intense electron sources with more
sensitive electron

detectors, and the use computer enhancement of noisy images, can be used to minimize the total energy
received by the sample.
• To reduce the possible dangers of artifacts, freeze-itching is used especially for the treatment of microbial
cells, unlike chemical fixation, dehydration, and embedding, where most specimens get contaminated.
• Microbial cell organelles undergo special treatment known as Freeze-itching whereby the specimens are
prepared with liquid nitrogen and then warmed at -100°C in a vacuum chamber.
• The sections are then cut with a precooled knife in liquid nitrogen at -196°C. After warming up the sectioned
specimen in a high vacuum for about 2 minutes, it can then coated ith platinum and carbon layer forming
replicas.
• These are then be viewed under the TEM displaying more detailed internal structures of the cell in 3D.
• This step of treatment with Liquid nitrogen is known as freeze-itching.
Applications of Transmission Electron Microscope (TEM)

TEM is used in a wide variety of fields From Biology,

Microbiology, Nanotechnology, forensic studies, etc. Some of
these applications include:
1. To visualize and study cell structures of bacteria, viruses, and
fungi
2. To view bacteria flagella and plasmids
3. To view the shapes and sizes of microbial cell organelles
4. To study and differentiate between plant and animal cells.
5. Its also used in nanotechnology to study nanoparticles such
as ZnO nanoparticles
6. It is used to detect and identify fractures, damaged
microparticles which further enable repair mechanisms of
the particles.
 It has a very powerful magnification of about 2 million times that of
the Light microscope.

It can be used for a variety of applications ranging from basic Biology

Advantages of to Nanotechnology, to education and industrial uses.

Transmission It can be used to acquire vast information on compounds and their

Electron structures.

Microscope It produces very efficient, high-quality images with high clarity.

(TEM)
It can produce permanent images.

It is easy to train and use the Transmission Electron Microscope

Limitations of Transmission Electron
Microscope (TEM)

1. Generally, the TEMs are very expensive to purchase

2. They are very big to handle.
3. The preparation of specimens to be viewed under the
TEM is very tedious.
4. The use of chemical fixations, dehydrators, and
embedments can cause the dangers of artifacts.
5. They are laborious to maintain.
6. It requires a constant inflow of voltage to operate.
7. They are extremely sensitive to vibrations and electro-
magnetic movements hence they are used in isolated
areas, where they are not exposed.
8. It produces monochromatic images, unless they use a
fluorescent screen at the end of visualization.
 Transmission Electron Microscope (TEM) Images

Transmission electron Transmission electron Transmission electron Transmission electron

micrograph of SARS-CoV-2 micrograph of SARS-CoV-2 micrograph of SARS-CoV-2 micrograph of SARS-CoV-2
virus particles, isolated from a virus particles, isolated from a virus particles, isolated from a virus particles, isolated from
patient. Image captured and patient. Image captured and patient. Image captured and a patient. Image captured
color-enhanced at the NIAID color-enhanced at the NIAID color-enhanced at the NIAID and color-enhanced at the
Integrated Research Facility Integrated Research Facility Integrated Research Facility NIAID Integrated Research
(IRF) in Fort Detrick, Maryland. (IRF) in Fort Detrick, Maryland. (IRF) in Fort Detrick, Facility (IRF) in Fort Detrick,
Credit: NIAID. Credit: NIAID. Maryland. Credit: NIAID. Maryland. Credit: NIAID.
Transmission electron micrograph of SARS-CoV-2 virus particles, isolated from a Transmission electron micrograph of SARS-CoV-2 virus particles, isolated from a patient.
patient. Image captured and color-enhanced at the NIAID Integrated Research Image captured and color-enhanced at the NIAID Integrated Research Facility (IRF) in
Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID. Fort Detrick, Maryland. Credit: NIAID