Dr. N.

Mohondas Singh
Associate Professor
Department of Chemistry
 Microscope:
The word microscope is derived from the Greek mikros (small) and skopeo (look at).
Microscopy involves the study of objects that are too small to be
examined by the unaided eye.
• The light microscope probably developed from the Galilean telescope during the 17th
century.
• One of the earliest instruments for seeing very small objects was made by the
Dutchman Antony van Leeuwenhoek (1632-1723) and consisted of a powerful convex
lens and an adjustable holder for the object being studied.
• With this remarkably simple microscope, Van Leeuwenhoek may well have been able to
magnify objects up to 400x; and with it he discovered protozoa, spermatozoa, and
bacteria, and was able to classify red blood cells by shape.
• The limiting factor in Van Leeuwenhoek's microscope was the single convex lens,
remedied later by the addition of a second lens, giving us the compound microscope--the
basis of light microscopes today.
• One hundred years later, in the 1920s, it was discovered that accelerated electrons
behave in a vacuum much like light.
• Furthermore, it was found that electric and magnetic fields could be used to shape the
paths followed by electrons similar to the way glass lenses are used to bend and focus
visible light.
• Ernst Ruska at the University of Berlin, along with Max Knoll, combined these
characteristics and built the first transmission electron microscope (TEM) in 1931, for which
Ruska was awarded the Nobel Prize for Physics in 1986.
 Electron Microscope
This electron microscope use
electron beam and magnetic
fields to produce the image
instead of light waves and glass
lenses used in the light
microscope.
Resolving power of electron
microscope is far greater than
that of any other compound
microscope. This is due to
shorter wavelengths of
electrons. The wavelength of
electrons are about 100,000
times smaller than the
wavelength of visible light.

The electron microscopes in the main suite are all fitted with digital image capture and
energy dispersive X-ray systems for analysis of elemental composition and
distribution.
100-250000 times than light
microscopy
 Elastic Electron Interactions
No energy is transferred from the electron to the sample
These signals are mainly exploited in
Transmission Electron Microscopy
Electron diffraction methods

Inelastic Electron Interactions

Energy is transferred from the electrons to the specimen
The energy transferred can cause different signals such as
o X-ray
o Auger electrons
o Secondary electrons
o Plasmons
o Phonons
o UV quanta or cathodoluminescence.
Used in Analytical electron Microscopy…. SEM, TEM…
Method:
The specimen to be observed is prepared as
extremely thin dry film [A thin film is a layer of
material ranging from fractions of a nanometer
(monolayer) to several micrometers in thickness. The
controlled synthesis of materials as thin films (a
process referred to as deposition)] is a fundamental
step in many applications) on small screens.
These are then introduced into the instrument at a
point.
The magnified image is viewed on a fluorescent
screen through an airtight window.
The image can be recorded on a photographic plate
by a camera built into the instrument.
Types:
There are two types of Electron
Microscope:
SEM (Scanning Electron Microscope)-
used to visualize the surface of
Objects.
TEM (Transmission Electron
Microscope) – allows one the study
of the inner structure.
Fig. simple block diagram of Principle of SEM and TEM

Fig. The interaction of

incoming primary electrons
with a sample. (a) Useful
signals
generated by
electronematter
interactions in a thin
sample. (b) Absorption of
SE, BSE, and
X-rays in thick samples, by
inelastic scattering within
the interaction volume,
limits the sample
depth from which they can
escape.
SEM:
It scans a focused electron beam over a surface to create an image. The
electrons in the beam interact with the sample, producing various signals
that can be used to obtain information about the surface topography and
composition.
Why use electrons instead of light in a microscope?
Given sufficient light, the human eye can distinguish two points 0.2 mm
apart, without the aid of any additional lenses. This distance is called the
resolving power or resolution of the eye. A lens or an assembly of lenses (a
microscope) can be used to magnify this distance and enable the eye to see
points even closer together than 0.2 mm.
Principle of SEM:
Accelerated electrons in an SEM carry significant amount of K.E., and this
energy is dissipated as a variety of signals produced by electron-sample
interactions when the incident electrons are decelerated in the solid sample.
These signals include secondary and back scattered electrons that produce
SEM images. The secondary electrons are emitted from the specimen
play the primary role of detecting the morphology and topography of
the specimen while the backscattered electrons show contrast in the
composition of the elements of the specimen.
 Instrumentation and Working Principle
• Electron gun consisting
of anode and cathode.
• The condenser lens
controls amount of
electrons travelling
down the column.
• The objective lens
focuses the beam into a
spot on the sample.
• Deflection coil (x,y scan
coil) helps to deflect the
electron beam.
• Secondary electron
detector (SED) attracts
the secondary
electrons.
• Additional sensors
detect backscattered
electrons and x-rays
Fig. Schematic diagram of the core components of an SEM microscope
Key Features of The SEM

• Electrons Gun: It contains filament and generates free electrons &

accelerate these electrons to energies in the range 1-40 keV in the SEM (In
low voltage SEM(LVSEM) lower electron voltage 1-5 keV is used; LVSEM gives
higher resolution of images).
• Vacuum: SEM is operated at low vacuum=>0.1 to 0.0001Pa & for volatile
sample; Variable pressure SEM(VPSEM) is used (pressure upto 4 kPa)
• Sample should be free from any contamination/dirt.
• If biological samples are used then any kind of water content should be
removed by suitable methods.
• Purpose of the electron lenses is to create a focused electron probe(to
search into and explore very thoroughly) on the sample.
• Most SEM can generate an electron beam at specimen surface with spot
size less than 10nm.
• Specimen: SEM can take specimens of up to 3-20 cm in diameter
depending on the specimen stage installed in the chamber.
• In order to produced images the electron bean is focused into fine probe,
which is scanned across the surface of the specimen with help of scanning
coils.
• With a higher accelerating voltage the electron beam penetration is greater
and the interaction volume is larger.
For SEM imaging samples are fixed in glutaraldehyde dehydrated through a series of solvents
and dried completely either by Air or Critical Point Drying method.
The sample/specimen is then mounted onto a special Metal Holder or stub and control with
a ultra-thin layer of Gold/Platinum/Carbon before image in SEM.
Condenser lens is used to narrow the electron beam given off by the electron gun (a lens
made of coils of wire that create an electromagnetic field which compress the electrons as
travel through it.)
Aperture ⇒allows controlling the diameter of the electron beam being passed through them
(blocks off any extra electrons that did not get fully condensed into the beam from hitting
the sample.)
Objective lens ⇒focus the electron beam down onto the sample (chamber-Vacuum). The
target itself needs to be conductive to prevent charging and allow for better image quality.
Thickness of the ultra-thin coating (sputter coating) is approximately 10 nm.
Coating is highly essential if the sample is beam sensitive or non-conductive.
Beam Sensitive Sample ⇒ Sputter coating (Ultra-thin coating) will prevent the damaging of
the sample by electron beam interaction.
Non Conductive Sample: Whenever electron beam will strike the sample surface, then
electrons of the beam will be trapped by the sample.
In the case of “Non Conductive Samples”; sputter coating will help to spread the
accumulated electrons in the surface.
Electron beam is controlled by anode, condenser lenses and objective lenses; there are
electromagnetic lenses.
Anode ⇒ is positively charged and attracts the negatively charged electrons; and ultimately
this anode will accelerate the velocity of the electrons.
 SEM
Detector

Secondary Back Scattered

X-ray detector
Electron (SE) or SE Electrons (BSE)
Detection
SEs are most abundant Inside the sample These are detected by
in the case of SEM and holder X-ray detector BSE detector and
detected by suitable is placed and it will again ET detector is
detector. detect the X-rays used for detection of
Everhart Thornley or ET emitted by the sample. BSEs.
Detector is most This detection process
commonly used is known as EDX or
detector for detection of Energy Dispersive X-
secondary electrons in Ray Analysis.
SEM.
 TEM SEM
Depth Resolution 1-10nm 10-100nm
Lateral Resolution 0.1-1nm 1-10nm
Magnification 200x-10,00,000x 10x-5,00,000x

Applications of SEM
SEM can measure and analyze the following
parameters:
Thickness of films and thin coatings
Surface morphology and appearance
Size and size distribution
Shape and dispersion of particles, fibers,
nanomaterials or any other additives in
composites and blends
Height and lateral dimensions of
nanometer-sized materials
Cell size and size distribution in foam
materials
Chemical composition and elemental
analysis of nano- and micro-materials
Fracture and structural defects analysis
Application:
It is well known that the
luminescence property of a
phosphor is strongly dependent
on its morphology and a
phosphor with spherical
morphology has more
advantage over the other
morphologies because spherical
shape phosphor gets more
brightness due to its high
packing densities and low
scattering of light. Fig. shows
SEM images for as prepared (a
& c) and 700◦C annealed (b &
d) samples of chosen prepared
compound recorded at low and
high resolutions. It is observed
from these images that the as
prepared sample exhibited
spherical shape with grain sizes
in the range of 200 nm to 1 m,
however incase of 700◦C
annealed sample, the particles
seems to be agglomerated and Fig. SEM images for room and 700◦C annealed as prepared sample
hence no particular shape have
been formed.
Advantages of SEM
They are easy to operate and has user-friendly interfaces.
They are used in a variety of industrial applications to
analyze surfaces of solid objects.
Some modern SEMs are able to generate digital data that
can be portable.
It is easy to acquire data from the SEM, within a short
period of time of about 5 minutes.

Limitations
They are very expensive to purchase
They are bulky to carry
They must be used in rooms that are free of vibrations and
free of electromagnetic elements
They must be maintained with a consistent voltage
They should be maintained with access to cooling systems
TEM (TRANSMISSION ELECTRON MICROSCOPE)
Introduction
TEM is a techniques for achieving high resolution images of thin samples/specimens. A
beam of high energy electrons passes through the specimens and is then focused to
form an image. The resolution of the TEM is greater than that of SEM, and is typically
of the order of 0.2nm. This compares with approximately 2nm for the SEM and around
0.2µm for the conventional optical microscope. Imaging in the TEM system must be
carried out under vacuum, electrons cannot travel through air.
It is a very powerful tool for material science. A high energy beam of electrons is shone
through a very thin sample, and the interactions between the electrons and the atoms
can be used to observe features such as the crystal structure and features in the structure
like dislocations and grain boundaries. Chemical analysis can also be performed. TEM
can be used to study the growth of layers, their composition and defects in
semiconductors. High resolution can be used to analyze the quality, shape, size and
density of quantum wells, wires and dots.
Basic principles:
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.
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.
TEM is the technique of choice for
analysis of specimen internal
microstructure, evaluation of
nanostructures such as particles, fibers,
and thin films, and imaging of atoms Fig.
2.8 shows the key components of a
TEM, which comprises the electron gun,
electrostatic lenses to focus the
electrons before and after the specimen,
and a transmitted electron detection
system.

Figure 2.8 Schematic of core components

of a TEM
Key features of TEM
Production of Electron Beam
Electron Gun: Tungsten filament/ LaB6 is used for production of electron beam in
electron gun. The electron gun in TEM typically accelerates electron through 80 – 300
kV accelerating voltage to give them sufficient energy to pass through upt to 1nm of
material. [In SEM, 1 – 30 kV].
200-300 keV electrons are typically used for routine imaging, with lower energy <100
keV electron used for analysis of very light elements such as carbon to reduce
specimen damage.
High accelerating voltage is applied for production of high intensity electron beam.
So, it can penetrate the sample up to the thickness of 1 micrometer.
OPERATING SYSTEM
TEM is operated under vacuum to reduce the any kind of interference(s) by air.
Anode ⇒ is positively charged and attract the electrons from the electron beam and
ultimately it will accelerate the electrons towards condenser lenses.
Condenser lenses ⇒ are electromagnetic lenses and they will focus all the electron
beam towards the specimen/sample. So, electron beam will be more accelerated and it
can easily penetrate the specimen. In TEM, number of condenser lenses are much
higher as compared to SEM.
Sample Holder in TEM is around 3 mm in diameter and up to 200 micrometer of
thickness.
Sample Method Preparation:

1. Fixation 2. Rinsing 3. Secondary Fixation 4. Dehydration

1. Fixation: i. Chemical fixation (Glutaraldehyde*)
ii. Cryofixation (Liquid N2 or He)

2. Rinsing: Sodium Cacodylate (Butter).

3. Secondary Fixation: For increasing the stability of internal structure
[OsO4]
4. Dehydration: Ethanol or acetone (suitable solvent)

Projector Lenses: It will magnify the transmitted beams coming from the
objective lens and focused towards the Fluorescence Screen or CCD.
CCD or Charged Coupled Detector will send the data to the CPU.

*Glutaraldehyde reacts with many nucleophiles in the cell (most commonly

amines). It produces irreversible cross-linking networks throughout the cytoplasm in
seconds to minutes. The reaction results in a drop in pH from a significant release of
protons, making adequate buffering important. Note that a too high concentration of
glutaraldehyde can inhibit the formation of rapid cross-links.
Cryofixation is a technique for fixation or stabilization of biological materials as the
first step in specimen preparation for electron microscopy and cryo-electron
microscopy.
TEM Imaging Mode
1. Bright Field Imaging (BFI)
2. Electron Diffraction Mode
3. High Resolution TEM (HRTEM)
4. Scanning TEM
5. High Angle Annular Dark Field (HAADF) TEM
Bright Field Imaging (BFI): Electron beam is passing through
specimen/sample, there are two possibilities – scattering and
unscattering.
Elastically Scattering: No loss of energy with same wavelength.
Inelastically Scattering: Loss of energy and changed angle.
Unscattered electrons are used for BFI. Actively scattering areas
of specimen will be showing darker contrast in BFI.
HRTEM: Electrons waves are interacting with crystal lattice,
diffract and forms complex interference pattern and visible at
40000X or more.
Scanning TEM (STEM): It contains scanning coils to scan the
specimen (across the specimen).
HAADF (High Angle Annular Dark Field): Incoherent and high
angle electrons are collected.
Applications:
TEM are capable of imaging at a significantly higher resolution than
light microscopes, owing to the small de Broglie wavelength of
electrons.

To examine fine detail-even as small as a single column of atoms,

which is tens of thousands times smaller than the smallest
resolvable object in a light microscope.

Application in biological sciences like cancer research, virology,

materials sciences as well as pollution, nanotechnology and
semiconductor research.

Application in chemical and physical sciences like in chemical

identity, crystal orientation, electronic structure and sample induced
electron phase shift as well as the regular absorption based
imaging.
The TEM image of as prepared
selected sample is shown in Fig. a
and it is seen from this figure that
the observed crystallites have
nearly spherical shape with sizes in
the range of 10–30 nm, which is
very close to the value of crystallite
size observed from XRD. A well
define lattice fringes have been
seen in the HRTEM image (fig. b),
indicating high crystallinity of the
sample and the calculated inter-
planar distance 0.302 nm
corresponds to the (211) plane of
as prepared sample lattice. The
SAED pattern (Fig.c) shows
concentric rings, indicating the
formation of polycrystalline.
Note: Peaks due to elements of
Cd, Mo, O and Sm have been
observed in the EDAX spectra of Fig. a) TEM image; (b) HRTEM image; (c) SAED pattern; (d)
as prepared sample shown in Fig. EDAX spectra of as prepared sample.
d, this result further supported the
dissolving of Mn+ into chosen oxide
lattice.
Advantages of TEM
It has a very powerful magnification of about 2 million times that of the
Light microscope.
It can be used for a variety of applications ranging from basic Biology to
Nanotechnology, to education and industrial uses.
It can be used to acquire vast information on compounds and their structures.
It produces very efficient, high-quality images with high clarity.
It can produce permanent images.
It is easy to train and use the Transmission Electron Microscope
Limitations
Generally, the TEMs are very expensive to purchase
They are very big to handle.
The preparation of specimens to be viewed under the TEM is very tedious.
The use of chemical fixations, dehydrators, and embedments can cause the
dangers of artifacts.
They are laborious to maintain.
It requires a constant inflow of voltage to operate.
They are extremely sensitive to vibrations and electro-magnetic movements
hence they are used in isolated areas, where they are not exposed.
It produces monochromatic images, unless they use a fluorescent screen at the
end of visualization.
Difference Between SEM and TEM
There are certain differences between a scanning electron microscope
(SEM) and transmission electron microscope (TEM), which are given as
below:
SEM detects scattered electrons emitted from the surface of the sample,
while TEM detects transmitted electrons.
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).
Both need electrically conductive materials to be tested. Non-conductive
materials should be coated with a conductive layer of metal or carbon.
The accelerated voltage ranges from 10 to 40 kV (kilovolt) for the SEM,
while for TEM, it is >100 kV.
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.
Contd.
The resolution is much higher in TEM as compared to SEM.
TEM is used for high magnification compared to SEM.
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.
SEM has a comparatively low resolution than TEM, while TEM has
high resolution.
SEM has a high depth of field, while TEM has moderate.
SEM has lower magnifying power, while TEM has high magnifying
power.
In SEM, the specimen contrast is by electron absorption, while it is by
electron scattering in TEM.
SEM produces three-dimensional black and white
References:
David B. Williams and C. Barry, Carter Transmission Electron Microscopy -A Textbook for Materials Science, Springer
Scienceþ Business Media, LLC 1996, (2009).
K. Akhtar et al. Scanning Electron Microscopy: Principle and Applications in Nanomaterials Characterization, (2019); DOI:
10.1007/978-3-319-92955-2_4
B.J. Inkson, Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for materials
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