An Assignment on

Structural and Functional aspects of differences between

Scanning and Transmission Electron Microscope

LS 202 Techniques in biology

Submitted To

Dr. Suchismita Das

Assistant Professor

Submitted By

Jyotirmoy Das

Roll No: 08

M.Sc. 2nd Semester

Dept. Life Science and Bioinformatics

Assam University Silchar

Introduction to Microscopy

Microscopy plays a crucial role in research and diagnostic aspects in the field of bioscience and
biomedical. With the advent of newer manufacturing processes and new technology, microscope is
playing a major tool for today’s biological scientist. This typically involves the use of optical
microscope for the analysis of microbial, cytological, pathological specimens for better contrast,
resolution and magnification[1]. It evaluates microscopic examination of various biochemical analyses
of samples for preparation of cells, tissues or organelle for an experiment to map the fine details of
spatial distribution of molecules within the cell. Magnification is indeed not the reliable measure to be
counted rather resolution defines the utility of the microscope to be able to analyze the distance of a two
closely points of a cell [2].

Over the long period, microscopy has undergone a renaissance with additional new technological
improvements. The visualization produced by microscope renders features of capturing the data into
recorded data electronically using computer imaging technology as digital camera acquisition, digital
image acquisition software, and digital display methods. Along with biological aspects of specimen
preparation, biomedical research have been improvised with advancements with microscopy
advancements[2].

Necessity of Microscope

The limitations of human eyes resolution power over the ability to determine two minimally distant
points below 1nm from each other applies the necessity for microscopy. Object below 1nm necessitates
the magnification by high end resolution microscope tool for studying and characterization to overcome
the limitations. The resolving power of the lens system depends on the ability of light to pass between
the objects being viewed. The resolving power, lens quality are not dependable for high output image
but also the wavelength of light[3]. The wave like forms such as radiowaves, X-rays and light make up
the electron magnetic spectrum, measured in nanometers. Limitations of physics of light and
possibilities of providing 10000x magnification necessitates EM which was one of the drawbacks of
OM.

Microscopes are categorized on the basis of source to produce image i.e by lenses, light wavelengths and
focused accelerated electron beam which is broadly classified into various optical and electron
microscope [4]. Electron microscope is again classified as scanning electron microscope and
transmission electron microscope.
Electron microscope

For visualization of ultrastructure imaging of cellular

structure, electron microscope is required which has
much higher resolving power than optical microscope.
The physical property of electron microscopes relies
specifically on preparation and staining techniques to
reveal the ultrastructure of cells and tissues. The
resolution of an electron microscopy depends on the
speed of the electrons in which higher the speed of the
electron, shorter the wavelengths and better the
resolution[5]. As electron possess shorter wavelengths
than visible light wavelengths, these electrons are much
energetic than light photons. Focusing of electron beam
on the surface of the material allows the electron to
surpass through the sample and scan materials on a fine
and very precise scale to create the image. As a result it
produces higher magnification and resolution
images[3]. Basic of an electron microscope
construction involves an Electron Gun, Anode,
Electromagnetic lens, Scanning coils, Specimen Holder or Stub, Detectors.

Structural and functional aspects of Scanning electron microscope (SEM)

Working Principle: Scanning Electron Microscopy

has played an essential tool in research areas since its
invention and has much significantly contributed towards
biomedical and life science fields[6]. It is a spectacular
technique as it provides a sense of depth it gives to
biological structures, thereby allowing surface
topography to be studied. The basic principle of SEM is
that it is based upon the interaction of an electron beam
with a specimen. It generates an image by scanning the
object with a beam of electrons[7]. When a high voltage of
between 40 000 and 100 000 volts (the accelerating
voltage) is passed between the cathode and the anode, a
tungsten filament emits electrons. The negatively charged
electrons pass through a hole in the anode forming an
electron beam. The beam of electrons passes through a
stack of electromagnetic lenses (the column). The incident
beam displaces orbital electron from the sample atoms,
giving rise to secondary electron emission. The number of
secondary electrons emitted depends on the specimen’s
electron density, which is related to its elemental
composition, so suitable detection of this secondary
emission yields information on the atoms of the surface
sample. Some primary electrons may not collide with orbital electrons. Instead they pass by the nucleus
to become backscattered electrons. These are not the only emission arising at the atomic level but these
two are the ones of interest for SEM operations[8]. In SEM, sample must be stained by element that
captures secondary e-.
Focusing of the electron beam is achieved by changing the voltage across the electromagnetic lenses.
When the electron beam passes through the specimen some of the electrons are scattered while others
are focused by the projector lens onto a phosphorescent screen or recorded using photographic film or a
digital camera. The electrons have limited penetration power which means that specimens must be thin
(50–100 nm) to allow them to pass through.
 Structural components
Electron Gun: This is located on the uppermost part of
the microscope. It consists of hair pin like tungsten
filament from which electrons are emitted upon heating
based on the necessity of material to get an image with
the best resolution with minimum sample charging and
damage. The function of the electron gun is to provide a
large and stable current in a small beam. The electron
emission source is mainly of, tungsten electron guns,
thermionic emitter, schottky, cold field emission, field emission gun. Electrons are produced by
thermionic emission or by cold field emission in which a fine tip of tungsten filament or lanthanum
hexaboride (LaB6) emitter is heated by an electric current flowing through the electron source to move
out the electrons[5]. The electrons in the filament possess a low efficient energy and therefore a high
voltage is passed between the electron source i.e. is cathode and anode to accelerate the electrons. In
cold field emission, the electrons are escaped from tungsten tip in room temperature. Its advantage is the
high yielding of electron without heating and low aberration of electron which allows imaging at atomic
resolution. These electron guns require high vacuum medium to function.

Fig: Electron Emitters[9]

Electromagnetic Lenses:

Electromagnetic lenses consist of windings of insulated copper wire, iron cast and pole piece. In the
lenses, electrons are gathered together by electromagnetic field. A magnetic field is induced by the
current in the electromagnets and the trajectories of the electrons on applying current on these coils
reach the pole of the lens. The electrons when enter the magnetic field gets deviated from the path
following the law of charge passing a magnetic field in which electron experience a force parallel to the
Z-axis, and this force will urge the electron to spiral through the lens. This spiraling causes the electron
to experience another force parallel to the radius of the lens, and this force compress the beam towards
the Z-axis. The electron lenses magnify or de-magnify the electron beam diameter, as their strength is
variable which results in a variable focal length.

Condenser Lens:

The electron beam on passing through the anode gets diverge from the emission source. By using the
condenser lens, the electron beam is converged and forms a parallel stream. A magnetic lens has hole in
the center of pole that allows the electron beam to pass through. A gap in the lens separates the two pole,
in which the magnetic field focuses the electron beam. The position of the focal point is controlled by
adjusting the condenser lens current. A condenser aperture is associated with the condenser lens. As
appropriate aperture size is chosen, many of the scattered electrons are excluded. A second condenser
lens is often used to provide additional control on the electron beam.

Scanning Coils
Scanning coils are used to scan the sample surface, and in order to get a higher magnification, current in
these coils is increased. The scanning coils deflects the electron beam above the object and with help of
this scan movement, the creation of the image occurs on the display. In case of slow scan, the refreshing
will be slow; the signal will be high while the noise will be low. On the other hand, if the scan is fast, the
refreshing will be fast, the signal will be low while the noise will be high. Scanning coils mostly contain
upper and lower coils, which avoid the creation of a circular shadow at low magnification.

Objective Lens
The objective lens is the lowest lens which focuses the beam on the specimen. Coarse focusing of the
specimen is done by choosing the working distance. Fine focusing can be subsequently done solely with
the objective lens. WD needs to be decreased for a specimen to be well focused. If the WD is less,
sample is at a higher position and close to the objective lens. Therefore, greater force is needed to deflect
the electron beam by objective lens. The shortest WD generates beam with the smallest diameter and
gives the poorest depth of field thus getting an image with the best resolution.
Secondary Electron Detector
The most widely used signal produced by the interaction of the primary electron beam with the
specimen is the secondary electron emission signal. When the primary beam strikes the sample surface
causing the ionization of specimen atoms, loosely bound electrons may be emitted and these are referred
to as secondary electrons. As they have low energy, they can only escape from a region within a few
nanometers of the material surface. So secondary electrons accurately mark the position of the beam and
give topographic information with good resolution. Because of their low energy, secondary electrons are
readily attracted to a detector called secondary electron detector.

Specimen Chamber
This contains the specimen holder and is situated at the base of the microscope column in the line with
the electron beam. Outside the specimen chamber, there are controls for movements of specimen X, Y
and Z direction and also for tilting and rotating. Both the column and chamber should be under vacuum.
There are separate diffusion pumps for column and specimen chamber and they are locked by a rotary
pump. As the electron beam hits the specimen surface, there is an interaction of electron with the
specimen. As a result of this, secondary electrons, back-scattered electrons, X-rays are produced, thus
there arises the possibility of several modes of imaging in SEM.

Sample preparation
Specimen preparation for SEM viewing is a lengthy process that must be carefully carried out. The three
dimensional effect on SEM-generated images is very useful for topographical studies. Usually low
voltage (~5 kV) is required to provide surface sensitivity. The sample can be probed to depths of tenths
of micrometers making it possible to convert a chemical structure into topographical relief. Samples
must be stable in the high vacuum environment. Mostly dry samples are used. Additionally, specimens
should be conductive, therefore a thin metal coating on the sample is needed. The remarkable depth of
focus allows the examination of uneven surfaces with the SEM. The analysis of elemental composition
of samples can be achieved by using additional detectors, especially for backscattered radiation[8].
 Structural and functional aspects of transmission electron microscope (TEM)

Working Principle: Transmission electron microscopy

works on the principle of transmitted electrons. TEM is
used to reveal sub-micrometre, internal fine structure in
solids. The TEM operates on the same basic principles as
the light microscope but uses electrons instead of light.
Because the wavelength of electrons is much smaller than
that of light, the optimal resolution attainable for TEM
images is many orders of magnitude better than that from a
light microscope. Thus, TEMs can reveal the finest details
of internal structure in some cases as small as individual
atoms. TEM depends critically on four parameters, the
resolving power of the microscope, the energy spread of
the electron beam, the thickness of the specimen, and the
composition and stability of the specimen[10]. In TEM,
electrons that pass through the specimen are imaged.

The specimen is bombarded by a beam of primary

electrons. The bombarding electrons are focussed to a
bundle onto the object. In areas in the object where these electrons encounter atoms with a heavy atomic
nucleus, they rebound. In regions where the material consists of lighter atoms, the electrons are able to
pass through. The fine pattern of electrons leaving the object, reaches the objective lens forms the
image. It is then greatly enlarged by projector lens. Eventually, the traversing electrons (transmission)
reach the plate at the base of the column of the microscope. The plate contains phosphorous compounds
that can absorb the energy of the stricking electrons and convert it to light flashes. Thus a contrasted
image is formed on this plate.
 Structural components

Electron gun
The electron gun generates the electron beam. It is usually positioned in
the top of the instrument column. In the image sequence below the gun
assembly is lifted off and moved aside to show how the electron emitter is
replaced. The emitter is seated within a cone-shaped Wehnelt cylinder
and the beam travels out of the small central hole apex of the cone.

Electron column
The electron column is made up of the gun assembly at the top, a
column filled with a set of electromagnetic lenses, the sample port and
airlock, and a set of apertures that can be moved in and out of the path
of the beam. The contents of the column are under vacuum. The
apertures can be easily removed from the beam path by the user. This
is important for operation of the objective and selected area
diffraction apertures during imaging. The apertures are located within
apertures strips, typically consisting of a strip of molybdenum
containing a sequence of different sized holes that allow modulation
of the beam to different degrees of precision. A cold trap which consists of a liquid nitrogen dewar
containing a conductive metal which is joined to a rod at the top. The rod penetrates the column and sits
near the sample. This cold area acts as a condensation site for material which leaves the sample. Such
material can contaminate the chamber or affect the vacuum status of the machine

Magnetic lens system

Within the column the electromagnetic lenses shape the electron beam,
which travels in a spiral trajectory. Each lens is constructed of a coil of
copper wire through which a current runs. There is a hole in the center
through which the beam travels. The inside of the column is maintained
under vacuum so that the density of molecules which can interfere with
the electron beam are minimised. To achieve this, systems of vacuum
pumps are attached to the TEM column.
Detectors
One of the most common detectors seen on a transmission electron
microscope is the x-ray energy dispersive spectroscopy (EDS or EDX)
system. This typically involves a large dewar for liquid nitrogen (to keep
the detector cold), an arm on which the equipment sits, and a solid state
detector that penetrates the column so it is located near the sample.

Specimen chamber
The specimen holder has one or two wells at the end. The sample is loaded
by a ring that screws into the well to hold it securely in place. The grid is
secured so that it does not fall out of the specimen holder. The holder is
then inserted into the column. During this process the sample airlock is
evacuated which can take a few minutes. To keep O-ring clean, the holder
is stored in a covering sleeve when outside of the machine. A cable can be
observed on some specimen holders. The cable is plugged into the column
to enable electronically-controlled tilting of the holder. This is important for obtaining precisely oriented
diffraction patterns and high resolution images.

Image capture
At the base of the column is a viewing chamber with a window port and an
adjustable pair of binoculars. The image is projected onto the screen in the
viewing chamber. The binoculars are available for focusing the image. The
screen in the chamber is only for producing a temporary image. To collect
a permanent image a CCD camera is inserted into the path of the beam.
This allows the image to be collected in a digital form. The length of time
that the beam is directed at the collection device can be adjusted to suit
beam parameters and to control the quality of the desired image.
 Sample preparation
A meticulous preparation of the sample is required for suitable TEM observation. This process is
lengthy and involves several steps, most of which are chemical processes: fixation, washing,
dehydration, infiltration with transitional solvents and with resins, embedding, and curing. Once the
specimen is chemically prepared it must be cut into extremely thin slices or sections. This procedure is
called ultramicrotomy and is performed to allow the beams of electrons to pass through the sample
material. Preparation of suitable thin samples for TEM studies in the area of biomaterials is hard to
accomplish due to the difficulty of performing ultramicrotomy, such as some polymers. There are some
alternative methods of specimen preparation that overcome this difficulty. However, this can be
overcome by using a defocused conventional TEM; a defocused beam also avoids sample damage or
destruction.[8]

Differences between SEM and TEM

There are certain differences between a scanning electron microscope (SEM) and transmission electron
microscope (TEM), which are given as below:

(i) SEM detects scattered electrons emitted from the surface of the sample, while TEM detects
transmitted electrons.

(ii) SEM provides information about surface morphology and composition of materials while TEM
gives details about internal composition of materials. Thus, TEM can illustrate several characters of a
material (morphology, crystallization, stress or even magnetic domains).

(iii) Both need electrically conductive materials to be tested. Non-conductive materials should be coated
with a conductive layer of metal or carbon.

(iv) The accelerated voltage ranges from 10 to 40 kV for the SEM, while for TEM, it is >100 kV.

(v) SEM requires a very easy preparation technique, while TEM needs skill to prepare a very thin
sample. The thickness of the specimen is not important in SEM, while specimen thickness is very
important in TEM. The thickness of the specimens to be examined under TEM should be less than 100
nm. SEM is a better tool for surface characterization as compared to TEM which is better for internal
structure analysis.

(vi) The resolution is much higher in TEM as compared to SEM.

(vii) TEM is used for high magnification compared to SEM.

(viii) In the case of SEM, the electron beam scans over the surface of the sample, while in the case of
TEM, the electron beam passes through the sample.

(ix) SEM has a comparatively low resolution than TEM, while TEM has high resolution.

(x) SEM has a high depth of field, while TEM has moderate.

(xi) SEM has lower magnifying power, while TEM has high magnifying power.

(xii) In SEM, the specimen contrast is by electron absorption, while it is by electron scattering in TEM.

(xiii) SEM produces three-dimensional black and white images, while TEM produces two-dimensional
black and white images.

(xiv) In SEM information can be obtained on a single viewing angle, while in TEM to obtain
information on the surface and near surface morphology, the viewing of the sample must be done at
several angles.
References

[1] G. McNamara, M. Difilippantonio, T. Ried, and F. R. Bieber, “Microscopy and image analysis,”
Curr. Protoc. Hum. Genet., vol. 2017, no. July, pp. 4.4.1-4.4.89, 2017, doi: 10.1002/cphg.42.
[2] K. Wilson and J. Walker, Principles and Techniques of Biochemistry and Molecular Biology, 7th
editio. Cambridge University Press.
[3] S. K. Sharma, D. S. Verma, L. U. Khan, S. Kumar, and S. B. Khan, Scanning Electron
Microscopy: Principle and Applications in Nanomaterials Characterization, no. February. 2018.
[4] S. Sheshrao Gajghate and by Sameer Sheshrao Gajghate, “Introduction to Microscopy A
Presentation on Introduction to Microscopy In-partial submission of PhD course work of
Advanced Instrument Analysis in chemical Engineering subject under Chemical Engineering
Department,” no. November, 2016, doi: 10.13140/RG.2.2.24105.49768.
[5] A. Kaech, “An Introduction To Electron Microscopy Instrumentation, Imaging and Preparation,”
Cent. Microsc. Image Anal., pp. 1–26, 2002.
[6] G. Chomette, M. Auriol, and J. M. Vaillant, “Scanning electron microscopy in oral cytology,”
Diagn. Cytopathol., vol. 2, no. 2, pp. 110–117, 1986, doi: 10.1002/dc.2840020204.
[7] M. P. Arora, Microbiology, 1st Edition. Himalaya Publishing House, 2005.
[8] J. G. Webster, Bioinstrumentation, Indian Edi. New York: John Wiley & Sons, 2004.
[9] A. Mohammed and A. Abdullah, “Scanning Electron Microscopy (Sem): a Review,” Proc. 2018
Int. Conf. Hydraul. Pneum. - HERVEX, no. January, p. 85, 2018.
[10] P. J. Goodhew, “General Introduction to Transmission Electron Microscopy TEM,” Aberration-
Corrected Anal. Transm. Electron Microsc., no. October, pp. 1–19, 2011, doi:
10.1002/9781119978848.ch1.