Microscopy is a means by which an object is transformed in to magnified image. There are different ways for magnifying the images of very small objects by large amounts. In any type of microscopy (optical microscopy or electron microscopy), a wave of wavelength λ (light wave or electron wave) interacts with the matter and as a result of this interaction we get the
microstructural information about the object. As the study of the materials at the nano-metric level is drawing much attention of the researchers in the current era, Electron Microscopy becomes a very important physical characterization tool at the nano-metric level. Electron Microscopy stands far ahead of the optical microscopy as it can provide the much improved
resolution and depth of focus compared to optical microscopy. This is a very introductory report on the basics of the electron microscopy (particularly on Transmission electron microscopy). Transmission electron Microscopy (TEM) operates on the same basic principles as the light microscope but uses electrons as “light source” and their much lower wavelength makes it possible to get a resolution thousand times better than with a light Microscopy.
3. INTRODUCTION
The microscope has been a fundamental tool for the research and study within different areas of
knowledge in which the human eye has no capabilities of presenting information about certain objects.
Approximately 1595 was a pivotal moment when Zacharias Janssen discovered by accident that when
a telescope’s tube is stretched out, a longer magnification could be achieved and thus allowing to
transform it into a microscope, since that moment many breakthroughs were being taken place as far
as microscopy techniques, however there was still an important barrier called “wavelength of visible
light”.
After De Broglie published his wavelength formula associated with the electron in 1924, Ruska
revealed that the wavelength related with the electron was five times less than the wave associated
with visible light, which implied and advance for the increase of the microscope resolution.
Between 1931 and 1933, Ernst Ruska acquire images of a metallic grid with electrons of 16X
magnification zoom, after that he got images of cotton fibers with 8x103X magnification zoom.
Electron microscopy can be considered as a technique that allows the attainment of specific and
special information about a sample or sample of study.
The transmission electron microscopy or TEM for its acronym in English, is a visual technique in
which an electron beam is focused on a sample to get an extended version of the image over a
fluorescent screen.
TEM and SEM are strongly used in areas of knowledge such as material science metallurgy and life
sciences research . magnification and of an aluminum sheet with 12x103X magnification.
4. TRANSMISSION ELECTRON MICROSCOPE
The transmission electron microscope (TEM) can be comprehended as a tool, artifact or instrument engineered
specifically for the analysis and visualization of samples or samples presented within ranges of dimensions
submitted from micro space of notation 1 × 10−6𝑚 (1 micrometer) and the nano space of notation 1 × 10−9𝑚 (1
nanometer).
This kind of electron microscope has capability of reveal highly complex levels of detail which are inaccessible
by conventional light microscope.
Figure 1 shows an image of a silver particles taken with the transmission electron microscope, note that as, these
nanoparticles are in the order of 50nm in length.
Figure 1. TEM image of Ag Nanoparticles monodisperse
5. To offer a more comprehensible notion of how the TEM substitutes the light optical microscope, an example can
be show in reference to the limited resolution in the observation of the bacterial cells morphology and extra large
viruses.
The dimensions of the bacteria range between 100 and 200 nm for the smaller ones, and up to 7µm in samples of
higher sizes; these can be observed using a light microscope, although to detail the subcellular bacterial
structures, it’s necessary to implement a TEM being that its largest dimension is less than 0.2µm, for this reason
a better resolution is achieved at the moment of replacing the light beam with the electron beam, which has a
wavelength of approximately 0.005nm.
TEM OPERATION
TEM is composed in the first instance by an electron source called gun or electron canon that usually is a V-
shaped filament made in 𝐿𝑎𝐵6 (lanthanum hexaboride) or in tungsten, an electric potential positive to the anode
is applied right here, which causes the filament (cathode) warms up until it produces an electron current with a
wavelength given by the De Broglie equation(1).
6. “Where 𝜆 is the wavelength
ℎ is the Planck constant
𝑚0 is the residual mass of the electron
𝑒 is the charge of the electron
𝑉 is the potential difference
𝑐 is the speed of light”
Before the electron beam reaches the sample, it is modified by the condenser and aperture lenses, in order to
improve the light coherence, that is to say, that the waves stay in same direction with a constant phase difference.
After this, the electron beam hits the sample where several processes are experienced in which the electrons that
affect the sample are dispersed avoiding the loss of energy (elastic) and other processes in which electrons hand
over part of their energy to internal electrons of the sample (inelastic).
Next, there is the objective lens which has the function of focusing the scattered beams to form the first image
thanks to a diffraction process performed by the projection lens which expands the electron beam and reflecting
in the phosphor screen.
The image resolution is a concept related to the amplitude and phase alterations in the electron beams, that is
caused by effects of the objective lens, this alteration is determined by the contrast transfer function (CTF) and
that is given by the equation (2)
7. Where 𝐴(𝑞)is a function that describes the diffraction diagram truncation by the aperture of the objective lens.
Exponential [𝑖𝑋(𝑞)] defines the phase function that describes the distortion of the output wave by the objective
lens.
According to this value, the contrast may differ, if the CTF is negative, the atoms will be shown with a black
contrast on a white background, on the other hand they will show a white contrast on a black background.
9. SAMPLE PREPARATION
The most important factor when preparing samples is that the technique used for this purpose does not affect the
sample to be observed, in TEM case, there are different types of techniques for sample preparation, but this
depends on the material and the information you want to obtain from it.
Mainly you can find two types of samples
1) self-supported
2) supported
For the preparation of self-supported samples, a procedure is performed to slenderize the sample, for this process
is necessary to obtain sheets of 100 and 200 nm thickness, and from these sheets are a cut to obtain a disc of 3
mm in diameter, this disc is polished by different forms that can be chemical, electrochemical, ionic
bombardment or ultra microtomy with the aim of reaching a thickness in the order of a few microns.
The supported samples are then found, which are usually deposited in a copper grid. The way to carry out this
process is to fragment the material in an agate mortar, then disintegrate in a part such as ethanol or acetone, then
one or two drops of the mixture are deposited on the grid, the solvent is allowed to evaporate and the sample is
introduced under the microscope.
10. LIMITATIONS AND IMAGE RESOLUTION
The TEM technique presents a series of drawbacks, one of the main ones is the consumption of a large amount of
time with a low efficiency in terms of the samples preparation, being that the thinning process for the samples to
being transparent to the electrons is quite extensive.
Another problem in terms about samples, is that you have to pay a large amount of money to observe a small part
of the sample.
The higher the resolution of the microscope, its sampling capacity deteriorates. For this reason, it is recommended
to observe the material with lower resolution techniques before implementing the TEM in order to have more
samples that offer other types of information.
Another of the most relevant problems of the TEM is that, this microscope throws 2D images of 3D samples, this
is a special care factor being that the human brain interprets the reflected light images.
If a complete characterization of the sample is desired, it is necessary to resort to techniques that are more
sensitive to depth, such as field ion microscopy, or Scanning Probe Microscopy.
11. On the other hand we can mention the effect that the electron beam has on the sample, since the ionizing
radiation is an effect that can damage it because of the high level of kilovolts; polymers and most organic
compounds are more susceptible to receive some kind of damage, for this reason it is of special care to work
with the microscope without having made routine tests of radiation leakage, since it can be harmful to health for
the high levels of radiation.
There are a number of limitations which cause the image resolution to have a series of restrictions regarding its
scope.
The image projected by the objective lens will be affected by an accumulation of aberrations such as spherical,
chromatic and astigmatism. "The spherical aberration consists in the reduction of the focal distance of the
electrons passing through the outer areas of the objective lens in regard to the electrons passing through the
center of it", this means that the rays that affect the Different lens distances do not converge at exactly the same
point. With the above, the following equation can be described:
12. Where 𝑑 is the smallest distance reflected in an image
𝜆 is the wavelength of the incident electron
Resolution can be determined as a limiting factor of the electron microscope.
Another limitation is found in the chromatic aberration, which is due to the variation of the refractive index with
the wavelength, and can be caused by fluctuations in the acceleration voltage, by the variation of beam energy of
emission, by loss of energies in the beam sample interaction or by alterations in the objective lens.
Finally, there is the astigmatism which occurs when the field of the objective lens does not have rotational
symmetry, despite this, four deflection coils placed after the objective lens are used to correct this problem.
13. APPLICATIONS
The transmission electron microscope (TEM) is a tool that works to analyze the structure, composition and
properties of samples in different areas of knowledge such as materials science, geology, environment, medicine,
electronics and biology in general.
Some of the main functions of the technique are:
1) Morphological analysis of small organism’s samples such as bacteria or viruses.
2) Inclusion of samples for subsequent collection and construction of three-dimensional images
3) Manipulation of samples while observing
4) Generation of X-rays for microanalysis
5) Analysis of the composition of the sample or of the atoms union states of the same one by means of electron
energy loss spectroscopy
Some of the most usual applications are, the determination of the crystallography of the phases, the
characterization of the defects of the crystal or the elemental analysis of ultra-small areas.
On the other hand, some of the more specific applications for which the TEM technique has been used are
described below.
1) Negative staining (contrast of samples) for the identification of viruses and bacterial compositions
2) Obtaining ultra-thin cuts
3) Characterization of ZnO nanotubes
4) Characterization of CdSe-graphene composite materials.
14. Next, a series of images taken from the transmission electron microscope are presented referencing some
applications.
Figure 3 shows the characterization of magnetic mesoporous silica Nano-particles, silica is the combination
of silicon with oxygen, which is found in some minerals, here we see the characterization of nanocrystals of iron
oxide, this characterization is done with the purpose of observing the interaction with proteins around these
Nano-particles so that they function as Nano-transporters with medical or pharmaceutical projections.
15. Figure 4 shows silver nanoparticles, these are used in electronics, clothing, paints, cosmetics, bactericides,
bio fungicides, biomedical applications, in the medical, pharmaceutical and food industry, in Figure 5-D the
SAED technique can be observed (Selected Area Electron Diffraction) which is a diffraction mode that uses an
aperture of the objective lens to determine the area that will contribute to forming a diffraction pattern of the
sample under the microscope. These patterns provide valuable information about the parameters of the crystal
lattice and the sample orientation.
16. CONCLUSIONS
The transmission electron microscope provides some advantages over the traditional optical
microscope, one of them is unlike the light microscope, which can offer results of approximately 200
nm, the TEM has capability to offer images resolutions up to 1 nm, which allows analyzing images
with magnifications of up to 1∗ 106 magnification zoom.
Thanks to this technique it is possible to calculate and observe the real structures sizes and that are
unreachable for an optical microscope.
Another thing that can be concluded with this description is that electrons cannot focus with glass
lenses like optical microscopes that uses light photons, for this purpose electromagnets are used in
order to focus the electrons towards the sample.
On the other hand, some limitations and disadvantages of the TEM technique are defined, among
which are the image resolution because of the spherical and chromatic aberrations of the lenses, there
is also the great consumption of time in the preparation of the samples to analyze.
In addition, you can see the presentation of 2D images, you can display a series of drawbacks when
interpreting the samples in 3D, so it is recommended to go to other techniques so you can get more
information and be more sensitive to the depth of the samples to be analyzed.
Finally regarding the limitations, it is recommended to take special care with radiation leaks. As for
the applications, in the TEM technique, you will find the analysis of bacteria or viruses, the
characterization of Nano-materials and the obtaining of ultra-fine cuts.
17. REFERENCE
Cristian Fabian Escalante Sierra, Fundamentals of transmission electron microscopy, the technique with the best
resolution in the world, February.2019
Dr. Rubén Omar Torres Barrera “A Simple illustration of transmission electron microscopy” Research in
Nanotechnology, Nanobiochemistry and Materials Physics. S.F.
Dr. Ioan Costina “Transmission Electron Microscopy (TEM)”, innovations for high performance,
microelectronics, S.F, p.1.
