Group 10: Electron Microscopy (TEM & SEM)

ADEBISI KAYODE E. 19/46ka150

Adesunloye Oluwafunke Tosin 19/46ka151

Adeyemi iyanu Elizabeth 19/46ka152

Akinlaja Judah Toluwanimi 19/46ka153

AYOBAMIDELE, Isaac Ayobami 19/46KA154

Bankole Susan Olubunmi 19/46ka155

Dada Tosin Ayodeji. 19/46ka156

James Onyinyechi Success 19/46KA157

OLABIYI DAMILOLA OPEADUA 19/46KA158

OLAWOYE, Faith-Blessing 19/46KA159

OMALE, Omanibe Johnson 19/46KA160

Onyemelukwe Chibuonu Johnpaul 19/46KA161

Osalohien Vanessa Ese 19/46KA162

Olumo Balqees Y. 18/55dz073

TAH Saundra AMUTENG 19/46Ka164

Introduction
Has we begin to learn more about the microscopic world using light microscopes that could
visualize individual cells, we started to become curious about structures that are smaller than a
cell, some details which were difficult to visualize and study under standard light microscopes.
Fortunately, electron microscopy developed in the 1930s and 1940s proved to be much more
powerful and allowed scientist to view these smaller structures. Some electron microscopy
techniques employ a resolution of 0.2nm which is more than 1000 times better than the light
microscopy. With this higher resolution, EM are able to magnify their specimen up to 1,000,000
times of the original allowing Researchers to study proteins inside of cells in the molecular
structures of viruses.
How is this achieved?
The answer is in the name. the beam of electrons interacts with the samples and the beam is
transmitted through the specimen. As recall from the Chemistry of electrons; they are stable
subatomic particles that carry a negative charge. Electron beams have a much shorter wavelength
than the visible light i.e., the light microscope use, which is referred to as De Broglie wavelength
at 0.01nm. This shorter wavelength allows for a high level of detailed resolution in imaging.
Types of Electron Microscopy
A. Transmission Electron Microscopy

 Parts of Transmission Electron Microscope:

Electron Gun: Produces electron beams using a tungsten filament cathode and a control grid.
Electrons pass through an aperture to the positively charged anode at high voltage.
Condenser lens system: focuses electron beam on the specimen.
Image-Producing System: Consists of objective lens, movable stage, intermediate, and
projector lenses.
Objective lens: produces intermediate image from the condenser.
Projector lenses: magnify the image.
Image-Recording System: Includes fluorescent screen for visualizing the monochromatic
image.
Digital camera: captures and records images, which can be stored in various formats.
Vacuum system: prevents electron-air molecule collisions and disruptions.

 Specimen Preparation:
Electron microscopes are run under high vacuum conditions. A high vacuum, however, is a very
unfriendly environment for biological specimens like cells and tissues (or condensed organic
hydrated matter in general). Electron microscopes pose the following prerequisites to the
specimen for investigation.
 Resistant in the vacuum
 Electron beams resistant
 Providing contrast
 Penetrable for electrons (transmission electron microscope)
Large biological specimens (cells, tissues) do not fulfill any of these requirements and have to be
treated in order to be investigated in the electron microscope. Typically, the specimen is turned
into a solid state to make it suitable for vacuum and resistant in the electron beam. Moreover, it
has to be cut to very thin sections of ca. 100 nm for transmission electron microscopy. Such a
specimen still does not provide contrast. Biological material is basically composed of light
elements like C, O, H, N, S, P, which do not interact with electrons. Selective staining or contrast
enhancement with heavy metals is required to form an image.

Types of Specimen Preparation for TEM

a. Classical Preparation for TEM
The classical preparation is performed at room temperature.
Fixation: the native biological specimen is chemically stabilized with chemical fixatives like
glutaraldehyde and osmium tetroxide. All biological processes are arrested during fixation.
Dehydration: specimen is ready for dehydration with a sequence of different ethanol
concentrations until it is completely dehydrated.
Embedding: the ethanol is exchanged with a monomer solution of a plastic formulation (e.g.,
Epon/Araldite) in a sequence of rising concentrations of plastic components dissolved in ethanol
or acetone. The monomers are polymerized by heat or UV light and provide a hard plastic block
containing the embedded specimen.
Thin Sectioning: This enables cutting of thin sections (100 nm) in an ultramicrotome or with a
diamond knife.
Staining: The sections are stained with uranyl-acetate and lead citrate. Uranium and lead ions
selectively bind to lipids, proteins and nucleic acid in the specimen and provide a distribution of
electron dense material
Mounting: a cover slip which is a thin piece of plastic or glass is used for this purpose.

NB: A lot of artifacts are introduced during classical TEM preparation. The fixation with
chemicals is slow (seconds to minutes) and the specimen has enough time to react to the
chemical attack and undergoes changes. The whole preparation procedure is conducted at room
temperature leading to extraction and shrinkage of material during dehydration and embedding
b. Cryo-Preparation/Cryo-fixation
Cryo-preparation methods were introduced and developed in order to prevent or reduce these
artifacts. In a first step, the specimen is generally frozen without chemical pretreatment (pure
physical process) – cryo-fixed. Freezing is very fast and can be performed within milliseconds.
However, the freezing is a challenging process because of the nature of water to form ice crystals
during freezing. Ice crystal segregations distort and damage the ultra-structure. High-pressure
freezing was developed to prevent ice crystal formation by changing the physical properties of
the water at 2100 bar just before freezing. Despite this technique, satisfying freezing quality
(Figure 6.3) is achieved only for biological specimens with a thickness of less than 200 µm.
Once the specimen is frozen, it is dehydrated and fixed with chemical fixatives simultaneously at
very low temperatures (e.g., -90°C, gradient to room temperature) in a process called freeze-
substitution. The embedding in plastic, sectioning, staining of the specimen is typically
performed the same way as during classical preparation.
For TEM specimens are fixed on a support grid and either embedded in resin or stained with a
particular compound like uranyl acetate and osmium tetroxide stain embedded in epoxy resin
(compounds of heavy metals). Certain structures within the specimen will take up more of the
stain than other parts of the specimen. Those sections are called electron dense region, when
electrons pass through electron dense regions, they lose more energy compared to when they
pass through less dense or sparse electron regions. This phenomenon is used by TEM to visualize
the specimen at high levels of resolution and magnification.
 Working Principles of TEM
When the specimen is secured in the specimen holder, a transmitted beam is generated and shut
out of the electron gun. The electron beam is accelerated down the column of the microscope
passing through the anode. The electron beam then passes through the specimen, losing energy
as the electron passes through electron dese regions, the electrons then pass through the objective
lens magnifying the image. Before the electrons pass through the projector or ocular lens, which
magnifies the image further and projects it unto the fluorescent screen. Then in transferring the
electrons into an image, the fluorescent screen is coated with a chemical which appears as bright
spot when electrons come into contact with it. The intensity of the bright spot depends on how
much energy the electrons have when they hit the fluorescent screen. The camera under the
fluorescent screen then captures the image made from the bright spot which is displayed on the
computer screen.
USES/APPLICATION: TEM are used to visualize structures that are too small to study with aid
of light microscope such as: cell organelles, proteins, cytoskeletal filaments and the component
of cell membranes.
LIMITATIONS: TEM is not suitable for the study of live specimens or image 3D structures
because, images must be dehydrated and cut into ultra-thin slices before imaging.
 B. Scanning Electron Microscopy (SEM)

 Parts of SEM

Electron Source: Generates the electron beam used for imaging.

Electron Optics: Focuses and directs the electron beam onto the sample/specimen
Sample Chamber: Houses the sample to be imaged.
Stage: Holds and positions the sample to be imaged.
Detectors: collect various signals emitted by the sample such as secondary electrons,
backscattered electrons, backscattered electrons, and X-rays.
Imaging System: Converts signals into an image on a display.
Vacuum System: Creates a low-pressure environment within the chamber to allow electron
beam propagation
Controls and Software: Allows users to adjust settings and manipulate images.
Scanning Coils: Control the position of the electron beam, enabling scanning over the sample’s
surface.
Aperture: Focuses the electron beam to a fine point.

 Specimen Preparation for SEM

 a. Classical preparation for SEM

Procedure for scanning electron microscopy involves critical point drying. Air drying of
specimens at room temperature leads to a collapse of the sample structure caused by the high
surface tension of water. Critical point drying was developed in order to prevent the destroying
forces of the surface tension.
Fixation: the native biological specimen is chemically stabilized with chemical fixatives like
glutaraldehyde and osmium tetroxide. All biological processes are arrested during fixation.
Dehydration: specimen is ready for dehydration with a sequence of different ethanol
concentrations until it is completely dehydrated.
The first steps including fixation and dehydration are performed the same way as described
for the classical preparation for TEM.
Clearing: After dehydration, the ethanol is exchanged with acetone. In a dedicated device
(critical point dryer) the acetone is exchanged with liquid pressurized carbon dioxide.
Critical point drying: subsequently, the temperature and the pressure in the chamber of the
critical point dryer are increased until they reach the critical point of CO2 (31°C, 74 bar). The
CO2 in the critical state (neither gas nor liquid) is released from the chamber very slowly
providing a gently dried specimen.
Dry coating: The critical point dried specimen is coated with a thin layer of heavy metal (e.g. 2
nm) by sputter coating or electron beam evaporation. The heavy metal layer renders the
specimen conductive and localizes the signal in the scanning electron microscope to the surface
providing the required yield of electrons and contrast for imaging.
b. Cryo-technique for SEM
Similar artifacts as during classical TEM preparation are introduced during this process since it
involves as well chemical fixation and dehydration at room temperature. Many of these artifacts
can be prevented again by using cryo-techniques for SEM (right path in figure 6.5). The freezing
or cryo-immobilization is the same as described for cryo-preparation for TEM. Once the
specimen is adequately frozen, it is fractured at low temperatures (e.g. - 120°C) in a dedicated
freeze-fracturing machine. The fracturing through the specimen is a random process providing
cross-fracture planes as well as fracture planes through biological membranes.
  Working Principles of SEM
Specimen is coated with vaporized gold or palladium ion because when electrons come into
contacts with coating the cause atoms on the surface of the specimen to emit electrons. These
immediate electrons are called secondary electrons. These are what SEMs use to visualize their
specimens. After a coated specimen is secured on the stage of the SEM in that an electron beam
is shut out and accelerated towards the beam of the specimen. The electron beam then scans the
surface of the specimen, which causes secondary electrons to discharge.
There are two types of detectors in the sample chamber:
1. Secondary electron detector
2. Backscatter electron detector
Backscatter electron detector back scatters electron that come from deeper regions of the sample
and that are a result of an interaction between the beam and the sample. The results from these
detectors are primarily used to look at crystallography and the magnetic field of the sample, but
for the purposes of microbiology, Researchers focus on the detection of secondary electron
which allows them to visualize the topography of the sample or the distribution of parts and
features on the surface of the sample.
The detectors convert the electron signals into a light signal that is amplified by passing through
a photomultiplier before the final image is projected on the computer screen
USES: SEM is primarily used to study the topography of cells and other similar structures
 SEM can produce 3D images opposed to the flat image (2D) from TEM
LIMITATION: Has worse resolution
Expensive and complicated to use

Electron Microscopy Advantages and Limitations

Advantages
1. High Resolution and Magnification: Shorter wavelength of electrons allows higher
resolution.
2. Nanoscale structures can be imaged with greater detail.
3. Imaging Subcellular Structures and Nanoscale Materials: Reveals intricate cell
organelles, aiding cell biology understanding.
4. Enables observation and characterization of nanomaterials.
5. Elemental Analysis using EDS: Coupled with EDS detectors for elemental analysis.
6. Detects characteristic X-rays emitted by electron-sample interaction.
7. 3D Imaging Capabilities (in SEM): Creates 3D reconstructions using stereo pair imaging
or electron tomography.
8. Visualizes complex surface structures.

Limitations
1. Sample Damage from Electron Beam: High-energy beams can cause radiation damage to
sensitive samples.
2. Vacuum Requirements and Sample Preparation: Samples need dehydration and
placement in a vacuum, altering characteristics.
3. Complex Sample Preparation: Thin sectioning for TEM can be technically challenging
and time-consuming.
4. High Cost and Specialized Training: Expensive instrumentation to purchase, maintain,
and operate.
5. Requires specialized training for proper operation.
6. Limited Sample Types and Electron Transparency: Samples must be thin and electron-
transparent, limiting observations.
7. Artefacts: Staining and fixation techniques can introduce artefacts.
8. Non-conductive samples may build up charge, causing distortions in SEM.
Applications
1. Biology and Medicine: Reveals cellular organelles, aids understanding of cellular
functions.
2. Facilitates study of virus and protein structures, aiding drug development.
3. Materials Science: Crucial for characterizing nanomaterials and understanding their
properties.
4. Enables study of crystal structures and defects.
5. Geology and Earth Sciences: Assists in mineral identification, contributes to geological
studies.
6. Nanotechnology and Electronics: Analyses semiconductor structures, ensures electronics
quality control.

Conclusion

In conclusion, electron microscopy stands as a cornerstone of modern scientific research,

unlocking a universe of previously hidden details within the microcosm. Its impact spans across
disciplines, from biology and medicine to materials science and beyond. As electron microscopy
continues to evolve, it holds the potential to unravel even more intricate mysteries of the
nanoscale world, pushing the boundaries of human knowledge and discovery.