Transmission electron microscope (TEM)

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 information is also detailed.
Early discovery of cathode rays like electrons by Louis de Broglie in the early 1920s,
paved way into the development of an electron microscope where they used a beam of
electrons creating a form of wave motion.
Magnetic fields were used as lenses for the electrons. With these discoveries, the first
electron microscope was later developed by Ernst Ruska and Max Knolls in 1931 and
modified into a Transmission Electron Microscope (TEM) by Ernst Ruska along with the
Sieman’s company, in 1933.
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.
Principle of Transmission Electron Microscope (TEM)
 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 TEM uses a beam of electrons to focus on the
specimen, to produce an image.
 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
1. Electron gun:
This is the part of the Transmission Electron Microscope responsible for producing electron
beams. Electrons are produced by a cathode that is a tungsten 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.

2.Image- Producing system

Its made up of the objective lens, a movable stage or holding the specimen, 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.

3. Image-Recording System
Its made up of the fluorescent screen used to view and to 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 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.
 Preparation of specimen for visualization by TEM
The specimen to be viewed under the TEM must undergo a special preparation technique to
enable visualization and creation of a clear image.
 Electrons are easily absorbed and easily scattered on solid elements, showing poor
visualization for thick specimens. And therefore, very thin specimens are used for
accurate and clear visualization forming a clear image as well. The specimen should be
about 20-100nm thin and 0.025-0.1nm diameter, as small as that of a bacterial cell. Thin
specimens allow interaction with electrons in a vacuumed space, are able to maintain
their innate structure.
 To get thin slice specimens, the specimen is first fixed on a plastic material with
glutaraldehyde or osmium tetraoxide. These chemical agents stabilize the structure of the
cell and maintain its originality. The addition of an organic solvent like alcohol such as
ethanol will dehydrate the cell completely for embedding the specimen to the plastics.
 The specimen is then permeated by adding an unpolymerized liquid epoxy plastic
making it hardened like a solid block. This is where thin sections are cut from using a
glass knife with a piece of special equipment known as an ultramicrotome.
 The specimen is then stained appropriately (with the appropriate stain) for the uniform
scattering of electrons. The thin sections are then soaked in heavy metallic elements such
as lead citrate and uranyl acetate allowing the lean and aluminum ions to bind to the cell
structures. This forms an opaque layer against the electrons on the cell structures to
increase contrast.
  The stained thin sections are then mounted on copper grids for viewing.
 The primary staining techniques that are applied for viewing under the TEM is Negative
staining coupled with heavy metallic elements coating. The metallic coating scatters
electrons which appears on the photographic film while uncoated sections and used to
study bacterial, viral cell morphologies and structures.
Freeze-itching treatment:
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.
Advantages of Transmission Electron Microscope (TEM)
1. It has a very powerful magnification of about 2 million times that of the Light microscope.
2. It can be used for a variety of applications ranging from basic Biology to Nanotechnology,
to education and industrial uses.
3. It can be used to acquire vast information on compounds and their structures.
4. It produces very efficient, high-quality images with high clarity.
5. It can produce permanent images.
6. 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.

High-resolution transmission electron microscopy (HRTEM)

High-resolution transmission electron microscopy (HRTEM) is an imaging mode of the

transmission electron microscope (TEM) that allows for direct imaging of the atomic
structure of the sample. HRTEM is a powerful tool to study properties of materials on the
atomic scale, such as semiconductors, metals, nanoparticles and sp 2-bonded carbon (e.g.
graphene, C nanotubes). While HRTEM is often also used to refer to high resolution scanning
TEM (STEM, mostly in high angle annular dark field mode), this article describes mainly the
imaging of an object by recording the 2D spatial wave amplitude distribution in the image
plane, in analogy to a "classic" light microscope.

What is HRTEM

HRTEM is an instrument for high-magnification studies of nanomaterials. High resolution

makes it perfect for imaging materials on the atomic scale. A main advantage of a TEM over
other microscopes is that it can simultaneously give information in real space (in the imaging
mode) and reciprocal space (in the diffraction mode). The instrument has a single tilt stage
and maximum Tilt Angle of -10º to +10o in Goinometer and the instrument can operate in
Bright-Field, Dark-Field, High resolution, SAED and CBED modes. It has a standard probe
and a variable temperature probe (100 to 500 K). TEM is coupled with a Gatan digital camera
for digital image processing.

Theory of operation

Basic principle of TEM is quite similar to their optical counterparts, the optical microscope.
The major difference is that in TEM, a focused beam of electrons instead of light is used to
"image" and achieve information about the structure and composition of the specimen. An
electron source usually named as the “Gun” produces a stream of electrons which is
accelerated towards the specimen using a positive electrical potential. This stream is then
focused using metal apertures and magnetic lenses called “condenser lenses” into a thin,
focused, monochromatic beam. Beam strikes the specimen and a part of it gets transmitted
through it. This portion of the beam is again focused using a set of lenses called “objective
lenses” into an image. This image is then fed down the column through the “intermediate and
projector lenses”, which enlarges the image, depending upon the set magnification. A
phosphor image screen is used to produce the image. The image strikes screen and light is
engendered, which enables the user to see the image. The darker areas of the image represent
the thicker or denser region of the sample (fewer electrons were transmitted) and the lighter
areas of the image represent those areas which are thinner or less dense (more electrons were
transmitted)

HRTEM can provide structural information at better than 0.2 nm spatial resolution. In most
crystalline inorganic materials, including ceramics, semiconductors, and metals, the positions
of individual atomic columns can be resolved, at least in low-index zones. When recorded
under optimum conditions, electron micrographs can be directly interpreted in terms of the
projected crystal potential. In other cases, image simulations are necessary to match proposed
structures to image features. Digital image recording and quantification of diffraction pattern
intensities is possible with the extreme linearity and high DQE of a CCD camera. Dynamic
events induced by the electron beam or indirectly with a heating holder can be followed by
video-tape recording from a TV-rate image pick-up system. At lower resolution, amplitude
contrast images can be used to observe material features in the 1µm-0.5nm range.
Possible Applications

 distribution and structure of defects, interfaces and grain boundaries

 nano-crystalline features in amorphous films
 small particle analysis in heterogeneous catalysts
 sub-micron morphological and device features
 thermodynamic decomposition, diffusion, and phase transformations

Limitations

High magnification imaging requires a high electron dose, so specimens need to be relatively
beam insensitive. The technique, by itself, provides very limited chemical information.
Heating experiments must be designed to minimize contamination of the microscope.