Electron Microscope
PREPARED BY:
BAHEZ O. ISMAEL
Introduction
• An electron microscope is a microscope that uses a beam of
accelerated electrons as a source of illumination.
• As the wavelength of an electron can be up to 100,000 times
shorter than that of visible light photons, electron microscopes
have a higher resolving power than light microscopes and can
reveal the structure of smaller objects.
• A scanning transmission electron microscope has achieved
better than 50 pm resolution and magnifications of up to about
10,000,000× whereas most light microscopes are limited
by diffraction to about 200 nm resolution and useful magnifications
below 2000×.
• It is a technique for obtaining high resolution images of biological
and non-biological specimens.
• EM s are used to investigate the ultrastructure of a wide range of
biological and inorganic specimens
including microorganisms, cells,large molecules, biopsy samples,
metals, and crystals.
Light microscope vs Electron microscope
“Types of electron microscope”
• There are two main types
of electron microscope:
1- The transmission EM
(TEM)
2- The scanning EM (SEM).
The transmission EM (TEM)
•The transmission electron microscope is used to view thin specimens (tissue sections,
molecules, etc) through which electrons can pass generating a projection image.
• TEM is used, among other things, to image the interior of cells (in thin sections), the
structure of protein molecules (contrasted by metal shadowing), the organization of
molecules in viruses and cytoskeletal filaments (prepared by the negative staining
technique), and the arrangement of protein molecules in cell membranes (by freeze-
fracture).
The scanning EM (SEM)
•Conventional scanning electron
microscopy depends on the emission
of secondary electrons from the
surface of a specimen.
•Because of its great depth of focus, a
scanning electron microscope is the
EM analog of a stereo light
microscope. It provides detailed images
of the surfaces of cells and whole
organisms that are not possible by
TEM.
Sample preparation for EM
• In order to be imaged using SEM, the specimen needs a
conductive surface and has to be placed inside high vacuum.
• Thus, biological specimens cannot be imaged in their native state
and need to be heavily processed.
•Accordingly, sample preparation is a crucial step.
•Materials to be viewed under an electron microscope may require
processing to produce a suitable sample. The technique required
varies depending on the specimen and the analysis required:
(Chemical fixation, Negative stain, Cryofixation ,Dehydration –
or replacement of water with organic solvents such
as ethanol or acetone, Embedding, metal shadowing, staining,
conductive coating by using gold, platinium or tungsten.
Sample preparation for topography
Sample preparation for topography
Step 1: Primary fixation with aldehydes (proteins)
Proteins are crosslinked by glutaraldehyde and formaldehyde to
stabilise the ultrastructure before further processing.
Step 2: Secondary fixation with osmium tetroxide (lipids)
Lipid membranes are fixed to prevent their extraction by solvents
during dehydration. The black osmium precipitate which is formed
during this process increases sample conductivity and minimizes
image distortions resulting from charging.
Step 3: Dehydration series with solvent (ethanol or
acetone)
A fixed specimen is dehydrated by incubation in a series of ethanol or
acetone solutions. Solvent concentration is increased gradually so
that water is removed gently, without causing specimen shrinkage.
Sample preparation for topography
Step 4: Drying
Allowing acetone or ethanol to simply evaporate from sample surface would
create artefacts as these solvents have relatively high surface tension and
would create micro-ripping of the surface upon leaving. To prevent this,
dehydration solvents are replaced either with Hexamethyldisilazane (HMDS)
or liquid CO2.
Step 5: Mounting on a stub
The specimen is mounted on a metal stub using a sticky carbon disc which
increases conductivity. Silver-containing glue can additionally be applied for
even more conductivity.
Step 6: Sputter coating with conductve material
To prevent charge buildup on specimen surface, it is coated with a
conductive material, most commonly gold. The metal is applied in a
controlled manner in a sputter coater. It is critical that the coating is thick
enough to prevent charging (typically around 10 nm) but not thick enough to
obscure specimen surface details.
Sample preparation for topography
Left: Three whole insect heads mounted on a stub and coated with gold, Right: One of the
heads imaged in the SEM
Sample preparation for topography
An insect coated in gold for viewing with Freeze-fracturing helps to peel open
a scanning electron microscope membranes to allow visualization of what is
inside
References:
• https://en.wikipedia.org/wiki/Electron_microscope#/media/File:Golde
n_insect_01_Pengo.jpg
• https://www.horiba.com/int/cathodoluminescence-spectroscopy-electr
on-microscope/
• https://www.britannica.com/technology/electron-microscope
• https://www.gu.se/en/core-facilities/sem-sample-preparation-techniqu
es
• https://en.wikipedia.org/wiki/Electron_microscope.
• https://www.acsmaterial.com/transmission-electron-microscope-tem.
html
Thank You
