Unit 1 Microscopy

UNIT 1
MICROSCOPY

Structure
1.1 Introduction Scanning Transmission
Electron Microscopy (STEM)
Objectives
Environmental Scanning
1.2 Imaging Techniques
Electron Microscopy (ESEM)
(Microscopy)
Comparative account of
Principles of Microscopy
different types of microscopes
Light Microscopy
1.4 Techniques for preparing
Phase Contrast Microscopy tissue for Electron Microscopy
Fluorescence Microscopy Negative Staining
Confocal Microscopy Freeze Fracture
Polarization Microscopy Freeze-Etching
1.3 Electron Microscopy (EM) Shadow Casting
Transmission Electron 1.5 Summary
Microscopy (TEM)
1.6 Terminal Questions
Scanning Electron Microscopy
1.7 Answers
(SEM)

1.1 INTRODUCTION
Cell biology is the branch of biology which deals with the study of cell
structure, their architectural details, major cellular as well as subcellular
processes, and the interactive processes between the cells. Various
techniques have been used to study the structural details and functioning of
the cell. Imaging techniques such as microscopy have proven very useful in
studying various aspects related to the architecture of cells, tissues and
various organelles present in them. The minute structural details of the cell,
cellular processes and other characteristics can be easily studied with the help
of microscopy techniques. The information procured via these techniques
provides a better understanding of the various cellular processes taking place
in a living being. Our knowledge about cellular structures and their functions
has improved manifold by using microscopy and biochemical techniques. In
this unit you will study about the various aspects of the microscopy techniques
and how microscopy helped to understand the structure of cell. An update
about various types of microscopy will also be provided to you. 9
Block 1 Techniques in Cell Biology

Objectives
Objectives
After studying this unit, you would be able to:

describe the structure, principle and functioning of a microscope in

detail;

enlist various types of microscopy techniques;

provide details about electron microscopy techniques;

enlist various types of electron microscopy;

describe the preparation of tissue for electron microscopy; and

describe the applications of microscopy techniques.

1.2 IMAGING TECHNIQUES (MICROSCOPY)

Various techniques are used to study the structural details of the cells and the
processes taking place in them. Microscopy is the most common technique
used to study the structural details of cells, tissues and organelles present in
plants and animals. A microscope is an instrument used to view small things
that cannot be seen with the unaided eyes. The word microscope is derived
from the word ‘micro’ that means small and ‘scope’ that means to look at. A
microscope magnifies objects many fold so that specimens can be seen
easily. The microscopy technique proves useful in visualization of cells and
microorganisms as the images appear much larger after magnification.

The most commonly used microscope is the optical or light microscope. In this
microscope the glass lenses are used to magnify the image.

History of Microscope

The concept of magnification came into existence long back in 1267 A.D. An
English philosopher R. Bacon invented eyeglasses in 1235 A.D. In his book
Perspectiva, he mentioned about the small size of dust and sand particles. In
1538, an Italian physician Girolamo Fracastoro observed that an object
appears much larger when seen with two spectacle glasses one
superimposed on the other. He mentioned these facts in his book
Homocentrica. The first simple microscope was invented by two Dutch
scientists, Zaccharias Janssen and his father, Hans in 1590. They were the
first to combine two concave lenses within a tube. They noted that nearby
objects appeared closer and larger. They developed a microscope made up of
wood and cardboard with an eyepiece and an objective lens. Antonie van
Leeuwenhoek, a Dutch microbiologist made first simple microscope in the
year 1668. He was able to see the presence of tiny living organisms present in
pond water. The organisms appeared like dots. His microscope was made up
of a double convex glass lens that was held between two silver plates (Fig.
1.1). Leeuwenhoek was the one who made microbiology popular throughout
10 the world.
Unit 1 Microscopy

Fig 1.1: An image of microscope used by Leeuwenhoek.

Single lens microscopes were first used by English natural philosopher Robert
Hooke in 1665 (Fig. 1.2).

(a) (b)

Fig. 1.2: a) The microscopes used in earlier times by Robert Hooke; b) image of
cover page of Micrographia.

Robert Hooke provided information about various microscopic objects (such

as fleas, lice, and nettles) in his book Micrographia. He also coined the term
‘cell’ for small, microscopic structures each living being is composed of.
Hence, invention of microscope resulted in the discovery of cell and ultimately
the formulation of Cell Theory. Dutch optician Francois Beeldsnijder,
developed an achromatic (non-colour-distorting) microscope objective in 1791.

In 1827, Scottish botanist Robert Brown demonstrated the presence of

nucleus. He coined the term ‘nucleus’ in 1831. English scientist Joseph
Jackson Lister designed microscope lenses free from achromatic aberrations
in 1830. Two single lens microscopes were commonly used in earlier times
(Fig.1.3).

In 1868, German physicist Ernst Abbe invented an apochromatic system of

lenses. He published lens theory in 1873. A British microscopist Julius
Rheinberg used colour filters in his microscope. The effect came to be known
as Rheinberg illumination in the19th century. The largest optical microscope
was Burch reflector made in 1947. The weight of this instrument was about
200 kg (440 pounds). The smallest microscope was made by British optician
Horace Edward Dall in 1950 that weighed no more than 24 grams (0.8
ounce). British doctor John McArthur designed commercially available small
microscope in 1958. 11
Block 1 Techniques in Cell Biology

Fig. 1.3: Images of two single lens microscopes used in older times.

1.2.1 Principles of Microscopy

A microscope is an instrument which contains two-lenses, a light source, and
mechanical adjustable parts. We need to be familiar with some terms such as
magnification, resolution, numerical aperture, illumination, and focusing before
studying in detail about the instrument. Magnification and resolution affect the
functioning of microscope to a great extent. The working of a microscope is
Magnification based upon the basic principle of magnification.
(enlargement)
Magnification - Magnification gives a measure of how much bigger an object
It can be calculated
by multiplying the appears under the microscope. In other words, it is the number of times the
magnification of the object is enlarged when observed under the microscope. You should know
objective used. that light microscope generally magnifies the object about 400 times the actual
Example- 10X
size. Magnification of the object depends upon the ocular lens and the
eyepiece, 40X
objective = 400X total objective lens.
magnification.
Resolution - A lens possesses the ability to show two adjacent objects as
distinct units. The smallest distance by which two points can be separated and
distinguished as individual objects is called resolution. The study of the
smallest detail of the object depends on the resolution. Resolution is
Numerical aperture
(NA) expressed in micrometres (µm), which is one millionth of a metre. Factors
such as wavelength of light used and the numerical aperture regulate the
NA= n sin α
resolving power of a lens. The diameter of the objective lens in relation to its
where
focal length is known as numerical aperture. The resolution of the microscope
n= refractive index of is determined by the numerical aperture (N.A.) of the objective lens.
the medium
Numerical aperture is also defined as the sine of half the angle of the cone of
α = half of the cone light taken by the objective multiplied by the refractive index (R.I.) of the
angle of light emitted
medium in which the object is immersed. Numerical aperture increases as R.I.
from the condenser.
increases. The values of N.A. of microscope having low-magnification
objectives range from 0.1 to 0.95 in case of dry objectives and 1.4 in case of
12 oil-immersion objectives.
Unit 1 Microscopy
Abbe gave the following formula for calculating resolving power of the
microscope.

d = λ / 2 NA

Where λ is the wavelength of light used in visualizing a specimen

NA is the numerical aperture. If using a green light of 514 nm and an oil

immersion objective with an NA of 1.45, the (theoretical) limit of resolution will
be 177 nm.

It has been noted that resolving power of the lens is higher if the wavelength is
short. Hence smaller the value of resolving power, higher the resolution and
more the clarity.

Box 1.1: Resolution

The resolution of an optical system is calculated using the formula

r = 0.61 λ/ n sin α
where r is the resolving power (It is the minimum distance between two points that
can be separated), λ is the wavelength of light used to illuminate an object, n is the
refractive index of the medium in which the object is placed, and sin α is the sine of
half the angle between the specimen and the objective lens. The term n sin α is
often referred to as the numerical aperture.

1.2.2 Light Microscopy

You might have used microscopes in the laboratories. These are called as
light microscopes or optical microscopes. In this type of microscope, visible
light passes through the specimen (the biological sample) and is bent to form
a magnified image. Lenses focus light on the specimen, magnifying it, thus
producing an image. The magnification varies depending on the types and
number of lenses used in the microscope. Optical microscopes are of two
types-simple or compound.

• Simple light microscope - A microscope having one lens is called a

simple microscope. This type of microscope can magnify an image up to
300X. It has low magnification as it uses a single lens. The resolution
power of a simple microscope is below 1 µm.

• Compound light microscope - A microscope having more than one

lens is called compound microscope. This type of microscope can
magnify images up to 2000X. It has a higher magnification as it uses two
sets of lenses, an objective lens, and an eyepiece. The lenses are
aligned in such a way that they can bend light for getting effective
magnification of the image (Fig 1.4). The resolution of compound
microscope is about 0.2 µm. The limitations of resolution (magnifying
power) found in a simple microscope can be overcome using this
microscope.

The magnification of the objective lens and eyepiece manage the magnifying
power of the compound microscope. 13
Block 1 Techniques in Cell Biology

Fig.1.4: A photograph showing a modern compound microscope.

Parts of a Microscope

A microscope mainly consists of many parts. These include an objective lens,

ocular lens, lens tube, stage, and reflector. An object is magnified through the
objective lens. When the target is focused, a magnified image can be
observed through the ocular lens.

Objective

The objective lens gathers light from the specimen, magnifies the image of the
specimen and projects this magnified image. Objective lenses show
magnifications in the range of 2 to 100X. Objective is designed to minimize
aberrations.

Objective can be of two types: dry or immersion. An objective which works

with the air between the specimen and the objective lens is called as dry. In
contrast, an objective that requires liquid and is usually transparent oil which
occupies the space between the object and the front element is called as an
immersion objective. In the first type of objective, refractive index (R.I.) of oil
matches that of the glass

The focal length regulates the optics of the objective. The focal length is
inversely proportional to the magnification. This means that focal length of an
objective decreases with the increase in magnifying power. .

In some objectives special glasses are used. These are called apochromats
and were first produced commercially by Abbe in the 1870s.

Aberration is noted in high-magnification objectives (magnifications greater

than 25X). Aberration influences the sharpness
sharpness or quality of the image. The
aberrations of a lens increase as the relative aperture of the lens is increased.

Eyepiece (Ocular Lens)

Eyepiece is the lens which is used to observe the image. These lenses are
usually of 10X or 15X magnification. The eyepiece helps in forming enlarged
14 virtual image that can be seen by the observer.
Unit 1 Microscopy
Body Tube

A hollow tube through which light travels from the objective to the ocular lens
is called as body tube (Fig. 1.5). It connects eyepiece to the objective. It
contains a prism that bends the light rays so they can enter the tube. The
upper end of body tube possesses ocular or eyepiece lens, while the lower
portion consists of a movable nosepiece containing the objective lenses.

Fig. 1.5: Diagram showing parts of a microscope.

Arm

It is a structure that connects head of the microscope to the base.

Base

The bottom of the microscope is called base.

Stage

It is a flat platform having clips that supports the slides (Fig. 1.4). In case of
mechanical stage, the slide kept in position with the help of two knobs. One
knob moves the slide left and right, the other moves it forward and backward.

Nosepiece

The circular structure that screws the objective lenses is called nose piece.
Objective lenses of varying magnification and numerical aperture are mounted
on the rotating nosepiece.

Condenser Lens

The condenser is mounted below the stage (where the specimen is placed)
and focuses beam of light onto the specimen. When it provides uniform
illumination in the region of the object under observation, it is referred to as
critical illumination. Alternatively, when the image is focused on the condenser
followed by objective of the microscope, it is referred to as Köhler illumination.
It is useful in getting sharp images of magnification 400X and above. For a
microscope with a power of 1000X or above, condenser lens with an N.A. of
1.25 or greater is needed. Such microscopes are generally equipped with an
Abbe condenser. 15
Block 1 Techniques in Cell Biology
Diaphragm or Iris

The diaphragm or iris regulates the amount of light entering the condenser i.e.
helps in adjusting the intensity of light. It is located under the stage or between
the light source and condenser.

IIluminator

A steady light source is used for illumination in the microscope. Mirrors were
used as a source of light in early microscopes. Mirror reflected light up from an
external light source located at the bottom of the stage. Generally, a tungsten
lamp is used as a light source. Effective illumination is required for getting
good magnification and resolving power. The amount of light entering the
microscope needs to be regulated as excessive illumination may obscure the
specimen.

Box 1.2: Refractive index

The resolving power of microscope depends on the refractive index (R.I.). Refractive
index is the capacity/power of light to bend while passing through air from the glass
slide to the objective lens. The refractive index of air is lower than that of glass. When
the light rays pass from the glass into the air, they bent or refract so that they do not
pass into the objective lens. Loss of refracted light can be compensated by mineral
oil which has the same refractive index as glass. The loss of light reduces the
numerical aperture and resolving power of the objective lens. The decrease in light
refraction produces more light rays which enter directly into the objective lens,
producing a clear image with high resolution.

Electronic detectors such as a complementary metal-oxide semiconductor

(CMOS) or charge-coupled device (CCD) chip is used to capture the
magnified image in modern day microscopes. The digital signal obtained is
transmitted to a computer and translated into an image on the monitor. The
pictures as well as moving video sequences are captured using software.

The specimen is mounted on a glass slide and immersed in a material which

has the same R.I. as that of the slide. The tissues are cut into thin sections
after treatment with fixatives. The preparations of this type preserve the
natural structural appearance of the cells. The acids and aldehydes such as
formaldehyde, acetic acid, and picric acid are widely used as fixatives. The
thin sections of the tissues are cut after getting them embedded in paraffin
wax or quick-frozen. The sections are cut using an instrument called
microtome. The thin sections are spread on the slides and stained. The
sample preparation is such that contrasts between the sub-cellular structures
as dark or light or coloured areas are produced. Contrast is produced by
applying specific stains (which give colour to different structures) or by
bringing alteration in light transmitting properties of cell components.

Box 1.3: Abbe theory of image formation in a microscope

The modern theory of image formation in the microscope was given by German
physicist Ernst Abbe in 1873. According to this theory, convergent light obtained from
a condenser helps in visualization of objects or specimens in the focal plane of the
microscope. A collection of plane waves propagating in a specified set of directions is
called as convergent light. The superimposition of waves produces incident
illumination. The diffracted waves are collected by the objective and directed to the
focal plane. Interference between the diffracted waves produces an image of the
object.
16
Unit 1 Microscopy
Abbe showed that more is the number of diffracted waves reaching the objective, finer
is the details of the image. The objective’s ability to collect diffracted light was
considered as the N.A. Greater is the magnification of the objective, greater is N.A. of
the objective.

Specialized optical microscope

The modifications and a range of special adaptations have been made in basic
optical microscope. In some specialized microscopes, the illuminator and lens
systems are incorporated into a single unit.
Inverted microscope
The light source and condenser situated in the upper part of the microscope
direct light down to the stage. The eyepiece in this instrument is angled
upward while the objective is set with its front element. The specimens present
in the liquid medium can be studied with the help of this microscope. The cell
cultures have been studied using this microscope. Hence the microscope has
proved useful in studies related to biology and medical research.
Stereoscopic microscopes (Dissecting microscope)
The microscope possesses two objectives and eyepieces. They provide
separate optical paths and different viewing angles to eyes. This arrangement
produces a three-dimensional visualization of the sample. Each eye can
independently image the object. The magnifying power ranges between 5 and
250X. This microscope is generally used for viewing the dissected subjects
and microsurgical procedures. They are also used in electronics
manufacturing units to monitor the bonding of leads to integrated circuits.

Metallographic microscope
In this type of microscope, vertical source of light (illumination) is used. The
light source is generally present below the eyepiece in the tube. Light is
focused through the objective onto the specimen. The reflected light or
scattered light gets back to the objective which is imaged back at the
eyepiece. This type of microscope is used to identify defects in metal surfaces,
determine crystal grain boundaries in metal alloys, and study rocks and
minerals. They also have applications in forensic science and diagnostics.
Reflecting microscope
Microscopes of this type possess reflecting objectives. An objective usually
consists of two components: a relatively large, primary concave mirror and a
smaller, secondary convex mirror which is located between the primary mirror
and the object. The objective lens of this type does not produce chromatic
aberration and needs no correction. The objects can be observed over a wide
range of visible light and also in the ultraviolet or infrared regions.
Brightfield microscope
Two lens systems are used for magnifying specimens. This includes ocular
lens located in the eyepiece and objective lens located in the nosepiece. The
beam of light obtained from tungsten lamp helps in illumination of the
specimen. The light is focused on sub-stage lens called a condenser. This
microscope provides a high-resolution image. These microscopes are used for
observing non-viable, stained samples. We cannot observe living cells
because of the absence of contrast between the specimen and the
surrounding medium. 17
Block 1 Techniques in Cell Biology
Darkfield microscope
In this type of microscope, the condenser is modified which directs light
obliquely on the specimen. This results in deflection or scattering of light,
hence making object appear bright against a dark background. This type of
microscope is suitable for observing living specimens.

Interference microscope
Two beams of light are used in this microscope. One beam of light passes
through the specimen while the other bypasses it. The combination of two
beams results in interference between them, thus revealing the structure of the
specimen. The difference between interfering beams of light is used to create
the image. This type of microscopy was developed by British microscopist
Francis Smith and French physicist Maurice Françon in 1947. Quartz lenses
were used by them to produce two beams of light that were perpendicularly
polarized.

1.2.3 Phase Contrast Microscopy

Phase-contrast microscope was invented by Dutch physicist Frits Zernike in
1934. He was awarded Nobel Prize in Physics in 1953. In this type of
microscope, a cone of light is passed through the specimen, which gets
diffracted. The light rays pass obliquely through the specimen, causing
refracted waves to deviate away from the unrefracted rays (Fig. 1.6). The
unrefracted and refracted rays are focused back into the image plane. Two or
more light waves combine to produce interference. This happens because
wave equal to the sum of the two combining waves is produced. The direct
and undeflected light creates a difference in phase producing contrast. The
phase difference of about one- fourth wavelength between two waves
produces only minimal interference.

Fig.1.6: Graphic explanation of principle of the phase contrast microscope. In

this microscope, delay in refracted rays by about one – fourth
wavelength than normal light microscope has been noted. A comparison
of the diagrams (on the right) illustrates that extra delay enhances the
interference obtained when the unrefracted and refracted rays are
18 focused back together (U+R).
Unit 1 Microscopy

The problem of staining and alterations in brightness can be overcome by this

microscopy. The technique proves useful for observing colourless specimens.
The transparent structure such as nuclei transmits a light when present in the
mounting medium that surrounds them. The propagation of light through object
produces a change in the optical path. The shift in the phase of light passing
through the structure and that passing around the structure forms the visible
image.

1.2.4 Fluorescence Microscopy

Fluorescence microscope is used to see tissues that fluoresce (absorb one
wavelength of light and emit another). Light of one wavelength excites the
fluorescent molecules while the light of other wavelength collects the emitted
light. Some cellular components fluoresce naturally and appear in various
colours, while non fluorescing structures fluoresce by staining with fluorescent
dyes (fluorochromes). Fluorescence dyes are used in preparation of
antibodies that bind to specific cellular proteins. The antibodies are first tagged
with fluorescein (a fluorescent dye) and then fluorescein – labelled antibody is
applied to cells (Fig. 1.7).

Fig.1.7: The network of microtubules present in this cell revealed using

fluorescent antibody labelling and fluorescence microscopy.

1.2.5 Confocal Microscopy

Confocal microscopy is an advanced microscopy technique which uses a
‘pinhole’ to eliminate out of focus light. The discrete optical sections can be
collected after eliminating out of focus light above and below the plane. High
power lasers illuminate the sample. They help in collecting light from the
desired focal plane. The beam of laser light gets scanned across the sample
and light only from the desired focal plane can enter the detector. This
microscope uses fluorescence optics. Fluorophores i.e. a fluorescent chemical
compound that can re-emit light upon excitation is used in the sample.
Fluorophores contain aromatic groups, planar or cyclic molecules with several
π bonds. When the laser light gets focused on a defined spot at a specific
depth located within the sample, fluorescent light gets emitted exactly at that
point. A pinhole cuts off out of focus signals from the optical path, thus
allowing the fluorescence signals from the illuminated spot to enter the light
detector and form image at different depths in the same sample (Fig. 1.8).The
technique is suitable for both live as well for fixed cells and tissues. 19
Block 1 Techniques in Cell Biology

Fig. 1.8: An image showing the working of a confocal microscope.

1.2.6 Polarization Microscopy

In polarization microscope, light source is equipped with a polarizing filter. The
linearly polarized light (whose wave travels in a single plane) passes through
the object. It either remains unaffected or splits into two beams with different
polarizations. The polarized light improves the contrast. Polarizer is a device
that transmits polarized light through the specimen. Analyzer is a device
placed above the objective. An analyzer fitted to the eyepiece blocks
polarization of the light. The analyzer is rotated to get maximum contrast in the
image and a difference in refractive index between planes is determined.
Polarization in the beam of light is removed by using objectives with low
numerical aperture. The differences in refractive index provide useful
information about the molecular substructure of cell organelles.

This microscopy is useful for visualising birefringent structures which differ in

refractive index in different planes. The crystalline substances, geologic
samples and fibrous materials such as the mitotic spindle and muscle fibers
can be easily studied using this microscope.

SAQ 1
a) Fill in the blanks with the appropriate word :

i) …………………….. provides measure of how much larger a

microscope causes an object to appear.

ii) Phase-contrast microscopy was invented by………………………. .

iii) ……………………. is a chemical compound that can re-emit

fluorescent light upon excitation.

iv) Illumination source in a phase microscope is a cone of light whose

20 rays pass …………………. through the specimen.
Unit 1 Microscopy
v) ………………….. is the instrument used to cut sections of tissues.

vi) In a ………………. microscope, visible light passes through the

specimen, gets bent through the lens system and forms the
magnified image.

b) State whether the following statements are ‘true’ or ‘false’ :

i) The confocal microscope uses fluorescence optics. Laser light is

focused onto a defined spot at a specific depth within the sample.

ii) In fluorescence microscopy, the light of one wavelength is used to

excite the fluorescent molecules, and the light of a different
wavelength is used for refraction.

iii) The advantage of a confocal microscope is that discrete optical

sections can be collected, eliminating the out of focus light above
and below the current plane of focus.

iv) Different organic compounds are used to preserve different

tissues.

v) Fluorescein (a fluorescent dye) labels antibody when applied to the

cells.
c) Write the functions of the following:

i) Eyepiece

ii) Diaphragm

iii) Body tube

d) Draw a neat and labelled diagram of a compound microscope showing

its different parts.

1.3 ELECTRON MICROSCOPY (EM)

Electron microscopy (EM) is a technique that helps in getting high resolution

images of specimens (biological and non-biological). A beam of electrons is
used as the source of illumination. The electrostatic and electromagnetic
lenses are used to control the beam of electron and focus it to form an image.

Basic Components of an Electron Microscope

1. The vacuum system creates strong vacuum in the entire column i.e.,
along the path of electron beam. This is required for making the
electrons travel as they do not travel easily in air.

2. Electron gun is used for emission of electron beam. Electron gun

consists of:

a) cathode which is a filament made of tungsten that emits electrons

at 50-100kV. 21
Block 1 Techniques in Cell Biology
b) anode that produces a powerful electric field. The hole in anode
provides an electron beam which makes the electrons travel at a
high speed (several hundred thousand kilometres per second).
The voltage applied is the range of 3-5 kilovolts (voltage is
required to draw electrons from the source).

3. Electromagnetic lenses are arranged together. They help in illumination,

focus, and magnification. The condenser lens controls the electron
beam, objective lens, intermediate lens and projector lens work together
to produce a final image (Fig. 1.9).

4. The photographic system is used to record image as an electron

micrograph.

5. A cooling system attached to the column prevents heating up of system

(since high voltage is used for emission of electrons).

This microscopy is widely used in biomedical research and study of detailed

structure of tissues, cells, organelles, and macromolecular complexes.

Fig. 1.9: Schematic diagrams of Light and Electron Microscopes showing

difference in the principle of working.

Electron microscopy is of two types- Transmission Electron Microscopy

(TEM) and Scanning Electron Microscopy (SEM). In TEM, the electrons are
transmitted through an object, focused by the lenses to form the image while
in SEM, electrons are reflected by the object to form an image.

1.3.1 Transmission Electron Microscopy (TEM)

The transmission electron microscope possesses high resolution. The concept
22 of electron microscopy was given by German physicist Ernst Ruska and the
Unit 1 Microscopy
electrical engineer Max Knoll in1931. The first functional electron microscope
was made by E.F. Burton and his students C. Hall, J. Hillier and A. Prebus
at the University of Toronto in 1938. Later in 1939, the first commercial TEM
was developed by Siemens.

In this type of microscope, a beam of highly focused electrons is directed

towards a thin section of the specimen (<200 nm). The highly energetic
incident electrons interact with the atoms in the sample and produce
characteristic radiation and particles which form image. A high voltage electron
beam is emitted by electron gun which is made up of a tungsten filament. The
cathode is used as the electron source while acceleration of electron beam is
produced by anode (Fig. 1.10). The electron beam is focused by electrostatic
and electromagnetic lenses. The transmission of electron beam through the
specimen results in magnification of the structure of the specimen through
objective lens. The transmitted electrons hit a fluorescent screen at the bottom
of the microscope and give rise to a "shadow image" of the specimen. The
image is projected onto a fluorescent viewing screen coated with a
phosphorescent or scintillator material. The photographic film or plate is
exposed directly to the electron beam or a fibre optic light-guide to the sensor
of a CCD camera. The image detected by the CCD may be visualized on a
monitor or computer.

The sample/specimen needs to be processed before viewing under an

electron microscope. The material/sample is prepared by different ways.
This is done either by cutting very thin sections of the specimen from a
piece of tissue after fixing it in resin or freezing it or isolating a specimen
and studying it after negative staining.

(a) (b)

Fig.1.10: a) Photograph of a TEM; b) Schematic representation of TEM showing

different electromagnetic lenses. 23
Block 1 Techniques in Cell Biology
Sample Preparation

As the TEM works under conditions of vacuum, water present in biological

materials needs to be removed. The tissue is preserved using different
fixatives to avoid tissue disruption caused by this loss of water. These fixatives
stabilize the macromolecular structure of specimen by cross linking
of proteins with aldehydes such as formaldehyde and glutaraldehyde,
and lipids with osmium tetroxide. The tissue is dehydrated using alcohol or
acetone and embedded before sectioning. The tissue is passed through a
'transition solvent' such as propylene oxide and then infiltrated with
an epoxy resin such as Araldite, Epon or Durcupan. The resin gets
polymerized (hardened) and the hardened sample is cut into very thin sections
using a diamond or glass knife present in the instrument called
ultramicrotome. The thin sections are picked up using a boat filled with water
made present around the glass knife. The cut sections float on the surface of
water which are picked up and placed on the surface of copper grid (Fig.
1.11). The staining is done with heavy metals such as lead, uranium, or
tungsten. The scattering of electrons produces contrast between different
structures. The specimens can be stained "en bloc" before embedding or later
after sectioning. In ‘en bloc’ staining, a fixed biological specimen (block tissue)
is immersed in an aqueous solution of uranyl acetatefor several minutes
and/or followed by aqueous lead citrate and/or lead aspartate. This process
enhances the image contrast of membrane structures or fibrous structures in
cells.

a b

c d

Fig.1.11: Image showing a) Knife Maker; b) Glass knife; c) Boats; d) Glass knife
with boat.

An Ultramicrotome

A microtome is a tool used to cut extremely thin sections of specimen/sample.

The word microtome is derived from Greek ‘micros’ meaning "small"
and ‘temnein’ meaning "to cut". it is also known as an ultratome or ultra-
microtome. The instrument is used in the preparation of ultrathin sections (50-
100 Å) that can be observed under TEM (Fig. 1.12). Glass and diamond
24 knives present in the instrument help in cutting very thin sections.
Unit 1 Microscopy

Fig. 1.12: Photograph of an Ultra-microtome.

TEM shows enhanced resolution but the electron beam used is very weak and
pass an appreciable distance through air, therefore high vacuum is required
inside the internal chamber of electron microscope. The photographs of cells
taken using an electron microscope are called electron micrographs. You
can see that the picture below shows the minute structural details of the
cellular entities (Fig. 1.13).

Fig. 1.13: Electron Micrograph showing two plant cells (X 45, 000). CW - Cell
wall; ER- Endoplasmic, M-Mitochondria, PM- Plasma membrane, PP-
Proplastids, T-Tonoplast.

1.3.2 Scanning Electron Microscopy (SEM)

In scanning electron microscope, electron beam moves over the specimen,
the molecules get excited and move to high energy level and emit secondary
electrons. These electrons are used to form image of the specimen. Image is
produced when electron beam is focused into an intense spot on the surface
of specimen. The spot is moved back and forth across the specimen by
charged plates called beam deflectors. These deflectors are located between
the condenser lens and the specimen. The signals sent by the deflector attract
or repel the beam. Secondary electrons are captured by a detector. The
scintillator emits photons of light on excitation when electrons are incident
upon it (Fig. 1.14). The deflected and emitted electrons are detected by a
photomultiplier tube. This tube forms a 3-D image of surface features of the
specimen. The electronic signal generated is transferred to the video screen. 25
Block 1 Techniques in Cell Biology

Fig. 1.14: Scanning Electron Microscope.

Generally, the image resolution of SEM is less than that of TEM. The SEM
image depends upon the surface processes. SEM can produce images of bulk
samples up to many centimeters in size. It produces images of three-
dimensional shape of the sample (Fig 1.15). This microscope has proved very
dimensional
useful in studying the minute structural details of the cell surface (Fig. 1.16).
The increasing popularity of SEM among cell biologists is because of its
remarkable ability to provide details
details about surface topography, show 3D
structure and improved resolution (30-100 Å).

26 Fig. 1.15: Schematic diagram of a SEM.

Unit 1 Microscopy

Fig. 1.16: Pollen structure of Cineraria plant showing spines as observed under
scanning electron microscope (image magnified 1,500X).

Sample preparation

The sample preparation for SEM involves various steps:

1. The material is fixed by immersing specimen in 2.8% glutaraldehyde

[prepared in 0.1M Hepes buffer (pH 7.2, having 0.02% Triton X-100)] for
several hours at room temperature or overnight at 4º C.

2. The fixed specimen is washed thrice (for 5 to10 min) in 0.1 M Hepes
buffer (pH 7.2).

3. The specimen is dehydrated by treating it with 25% ethanol for 10 min,

50% ethanol for 10 min, 70% ethanol for 10 min, 85% ethanol for 10 min,
95% ethanol for 10 min, and 100% ethanol for 10 min.

4. The automated process of Critical Point Drying is carried for 40 min.

5. The sample is mounted on metal stub with double sided carbon tape.

6. The sample is coated by applying a thin layer of metals (gold and

palladium) using an automated sputter coater.

1.3.3 Scanning Transmission Electron Microscopy

(STEM)
Scanning Transmission Electron Microscope combines elements of both TEM
and SEM. STEM is a sophisticated technique and requires high vacuum
conditions. It is electronically more complex than TEM or a SEM (Fig. 1.17 a).
STEM is equipped with scanning coils, detectors and necessary circuitry. The
image is formed by the secondary electrons since the primary electrons
transmit light across the specimen. The electron beam is focused to a fine spot
(size 0.05 – 0.2 nm) which is then scanned over the sample. The magnetic
lenses focus beam of light that seeps over the surface of the specimen (Fig.
1.17 b). The sample is illuminated at each point with the beam parallel to the
optical axis. The technique of STEM helps in distinguishing specific
characteristics of the electron transmitted by the specimen, thus providing
unique information about the specimen. 27
Block 1 Techniques in Cell Biology

(a)

(b)
Fig. 1.17: a) Scanning Transmission Electron Microscope (STEM); b) Schematic
diagram of STEM.

1.3.4 Environmental Scanning Electron Microscopy

(ESEM)
Environmental Scanning Electron Microscope (ESEM) is a specialized SEM in
which uncoated specimens are analysed. The first commercial ESEM was
28 produced by the Electro Scan Corporation in USA in 1988. The instrument
Unit 1 Microscopy
produces high quality and resolution images in wet samples and those
contained in low vacuum or gas (Fig. 1.18). The samples present even in low-
pressure gaseous and high humidity (up to 100 %) conditions could be
observed clearly. The technique enables the imaging of biological samples
unstable in high vacuum conditions (conventional electron microscopes). This
is because this microscope possesses a secondary-electron detector which is
capable of operating in the presence of water vapour. The pressure-limiting
apertures separate the vacuum region from the sample chamber. The
specimen is placed in a relatively high-pressure chamber and the electron
optical column is differentially pumped to keep vaccum adequately low at the
site where the electron beam is exposed.

Fig. 1.18: Environmental Scanning Electron Microscope (ESEM).

ESEM is useful for studying non-metallic and biological materials (Fig. 1.19).

Fig. 1.19: Structure of epidermis with stomata as seen in ESEM.

1.3.5 Comparative Account of Different Types

of Microscopes

Each microscope shows variations in its structure, technical details and

produces images of characteristic type. The details about various microscopic
techniques have been provided to you in the sections above, a summary of
comparative account of the characteristics of different microscopes has also
been given to you for more clarity (Table 1.1 and Fig. 1.20). 29
Block 1 Techniques in Cell Biology
Table 1.1: Comparative account of different types of microscopes.
Characteristics Compound Confocal Scanning Transmission
Microscope Electron Electron
Microscope
microscope Microscope
(SEM) (TEM)
Source of Light Laser light Electrons Electrons
illumination for
image formation
Type of Lenses Glass Glass with One One electrostatic
dichromatic electrostatic and and few
mirrors few electromagnetic
electromagnetic lenses
lenses
Medium of image Air Air Vacuum Vacuum
formation
Sample preparation Sample Sample The specimen is Thin sections of
preparation is preparation is coated with gold the specimen are
not required not required used.
Specimen mounting Glass slides Glass slides Aluminum stubs Coated or
with dyed uncoated copper
samples grids
Focusing, Changing Digitally Electrical Electrical i.e.,
Magnification and objectives enhanced changing current
Adjustment of the projector
lens coil
Means for obtaining Absorption of Laser light with Electron Scattering of
specimen Contrast light dichromatic scattering electrons
mirror
concentrated
at pinhole
Type of Image Two- Three- 3-D image is 2-D view image
obtained dimensional dimensional seen is formed.
image image.

(a) (b)

(c) (d)
Fig. 1.20: Image micrographs of an algal cell as seen under different
30 microscopes. a) Compound; b) Confocal; c) SEM; d) TEM.
Unit 1 Microscopy

SAQ 2
a) Match the items in column l correctly with those of Column ll.

Column I Column II

i) Confocal microscope 1. invented by Manfred von Ardenne.

ii) SEM 2. uses electron as the source of

illumination and 3-D images is formed

iii) TEM 3. uses laser light as the source of

illumination

iv) Ultra-microtome 4. useful for non-metallic and biological

materials

v) ESEM 5. used for the preparation of ultrathin

sections

b) Fill in the blanks with appropriate words

i) In an electron microscope, a strong ……………… is required for

the travel of path of electrons.

ii) A ……………… system is attached to the column in an electron

microscope to prevent heating of system due to high voltage of
electrons.

iii) In a transmission electron microscope (TEM), electron gun made

up of ……………… filament cathode acts as the electron source.

iv) ……………….. is a more sophisticated and electronically more

complex technique than TEM or SEM.

v) A specialized SEM in which microanalysis can be performed on

uncoated specimens is ………………. .

1.4 TECHNIQUES FOR PREPARING TISSUES

FOR ELECTRON MICROSCOPY
Technique of electron microscopy helps in characterizing the microstructural
features of a sample ranging from 100 nm to 100 µm. Various methods
pertaining to processing/preparation of samples have been developed over the
years.

1.4.1 Negative Staining

Negative staining method is used for examining very small objects. It is
generally used in TEM. The shape and surface characteristics of intact
organelles or viruses can be examined using this technique. In the method,
thin sections are not cut but particles are suspended in a small drop of liquid
applied to copper grid and allowed to dry in air. The dried surface of sample is
treated with stain such as phosphotungstic acid or uranyl acetate (Fig. 1.21).
The specimen is visualized against the stained dark background. 31
Block 1 Techniques in Cell Biology

NEGATIVE STAINING POSITIVE STAINING

Fig 1.21: Electron micrograph of negatively stained wound-tumor virus (left) and
positively stained collagen fibers (right).

1.4.2 Freeze-fracture
The technique involves physical breaking (fracturing) of a frozen biological
sample along the planes of natural weakness that run through each cell.
These planes generally occur between the two layers of lipid molecules which
forms part of membrane and around various organelles of the cell. The main
steps involved in formation of a freeze-fracture replica are; (i) rapid freezing,
(ii) fracturing, (iii) formation of replica, and (iv) replica cleaning. The
pretreatment carried out before freezing comprises fixation of material in
glutaraldehyde followed by cryoprotection with glycerol. Cryoprotection is done
to reduce the formation of ice crystals during freezing. This kind of sample
preparation provides planar views of the internal organization of membranes.
Images provided by these samples have proved useful understanding the
functional morphology of the cell. This technique has been used to study
membranes and reveal the pattern of integral membrane proteins.
General outline of technique:

i) Cells are frozen in liquid Freon (-130º C). This results in instant
immobilization of cell components.

ii) The block of frozen cells is fractured in an evacuated chamber. The

fracture is irregular and occurs along lines of weakness like the plasma
membrane or surfaces of organelles.

iii) A replica of the fractured specimen is made by shadowing with platinum

(Fig. 1.22).

iv) A carbon replica is produced when a thin layer of carbon is evaporated

vertically onto the surface.

v) Replica is left behind after digestion of organic material by acid.

32 vi) The replica is studied under EM.

Unit 1 Microscopy

i)
ii) iii)

iv) v) vi)

Fig. 1.22: Diagrammatic representation of various steps of freeze fracture

technique (i-vi).

1.4.3 Freeze-Etching
The freeze-etching technique of sample preparation is related to freeze
fracture. One more step is added to freeze fracture procedure. In this method,
the specimens are rapidly frozen in liquid Freon under vacuum and struck with
a sharp knife edge as in freeze fracture method (Fig. 1.23).

(a)

(b) (c)
Fig. 1.23: Freeze fracture views of organelles a) entire cell showing nucleus and
nuclear pores; b) Golgi apparatus; c) Mitochondria. 33
Block 1 Techniques in Cell Biology
Biological samples fracture along lines of natural weakness at low
temperature. The tissue is subjected to vaccum which results in sublimation of
water from the fractured surfaces. This removal of water produces an “etching”
effect. This etching will cause small areas of the cell surface around the
periphery of the fracture face to stand out against the background. A replica of
the specimen is made by heavy metal such as platinum, followed by backing it
with a carbon film. After dissolving the tissue in strong acid, the remaining
metal replica can be viewed with the electron microscope. Such preparations
provide a unique picture of cells. This procedure is useful because it avoids
exposure to fixatives, embedding agents, and stains which can deform
ultrastructure of cell (Fig. 1.23). This type of specimen treatment causes
minimal tissue distortion and permits immediate arrest of cell function

1.4.4 Shadow Casting

Shadow-casting is a technique which helps in studying surface texture of the
specimen/material. The method involves in vacuum deposition of a metallic
film on dried specimens. A three-dimensional electron beam image is
produced using this technique. This process involves deposition of an electron
dense substance upon the specimen at oblique angle so that only a part of the
specimen is coated (Fig. 1.24). The deposition of metal on one side creates a
“shadow” resulting in three-dimensional appearance.

Fig. 1.24: a) Technique of shadow casting; b) Vacuum evaporator.

34
Unit 1 Microscopy
The thin metal film is formed on the specimen by condensation after
vaporization. The metals with the high vaporization temperature condense
quickly after vaporization (Fig. 1.25). The isolated particles can be visualized
by placing them in an evacuated chamber and spraying heavy metal across
their surfaces. The particle size of a film of evaporated gold will be larger than
that of platinum or palladium. The "grain" size of evaporated tungsten is
exceedingly fine, but its deposition time is long. The concurrent evaporation of
two or more elements results in smaller aggregate size by increasing the
distance over which any atom diffuses within a crystal lattice.

Fig. 1.25: Electron micrograph of Tobacco mosaic virus shadowed with

uranium.

SAQ 3
a) Answer in one word:

i) The process in which two or more two or more light waves

combine to reinforce or cancel one another, producing a wave
equal to the sum of the two combining waves.

ii) The other name for Bright field microscope.

iii) A fluorescent dye used for staining the microscopic samples.

iv) Focused beams of laser light are scanned across the sample and
light only from the desired focal plane can enter the detector.

v) This type of microscopy is useful for visualising birefringent

structure, which differ in refractive index in different planes.

vi) In TEM technique, these fixatives stabilize the specimen's mobile

macromolecular structure by chemical cross linking of protein.

vii) The photographs of cells taken using an electron microscope.

viii) The technique consists of physically breaking apart (fracturing) a

frozen biological sample along the planes of natural weakness that
run through each cell.

b) Enlist major differences between Freeze fracture and Freeze etching

techniques. 35
Block 1 Techniques in Cell Biology
1.4 SUMMARY
• Cell biology is the branch of biology that provides information about cell
structure and function. A combination of microscopic and biochemical
techniques has enhanced our knowledge and understanding of cell
structure and function.

• The discovery of microscope has proved as a milestone in the study of

cell biology as minute structural details and major cellular mechanism
could be revealed by this technique.

• Initially, light microscope proved very useful in studying the structural

details of cells, though lower resolution (200 nm) restricted the study of
detailed cell architecture.

• Various other versions such as phase-contrast, confocal and

fluorescence microscopes developed over the time provided a medium
of studying structural details of the cell/sample/specimen present in any
medium or form.

• The change of source of illumination from light to electrons significantly

improved the resolving power of the microscope. The invention of the
transmission electron microscope improved our understanding of cell
biology but the sample to be viewed required preparation before use.

• Various advancements such as Transmission Electron Microscope

(TEM) and Scanning Electron Microscope (SEM) proved very useful as
they showed remarkable ability to study surface topography and 3D
structure, along with improved resolution (30-100 Å). The electrons are
transmitted through an object and then focused by the lenses to form the
image in TEM while, the electrons are reflected by the object in a
scanned pattern which forms the image in SEM.

• Scanning Transmission Electron Microscope (STEM) contains elements

of both TEM and SEM. It provides unique information about the
specimen. STEM requires high vacuum condition and electronically
more complex than a TEM or a SEM. This microscope is equipped with
additional scanning coils, detectors and necessary circuitry.

• A specialized SEM, Environmental Scanning Electron Microscope

(ESEM) performs analysis using uncoated specimens. The samples are
observed in low-pressure gaseous and high relative humidity (up to 100
%) environments. It produces quality images and gives good resolution
even for wet samples or those contained in low vacuum or gas.

• Diverse set of procedures for specimen preparation, such as thin

sectioning, negative staining, positive staining, shadow casting, whole
mounting, and freeze-fracture has been developed so that minute
architectural details, three-dimensional view of the cell surface can be
36 captured, and details of the cellular processes can also be revealed.
Unit 1 Microscopy

1.5 TERMINAL QUESTIONS

1. Define the following terms
a) magnification
b) resolution of microscope
c) ESEM
d) STEM
e) negative Staining
f) shadow Casting
2. Discuss the factors which determine the resolving power of a
microscope.
3. Describe various types of electron microscopes.
4. Differentiate between
a) simple and compound microscope
b) TEM and SEM
5. Enlist the differences between confocal and fluorescence microscopy.

1.5 ANSWERS
Self-Assessment Questions
1. a) i) magnification
ii) Frits Zernike
iii) fluorophore
iv) obliquely
v) microtome
vi) light
b) i) True; ii) False; c) True; d) False; e) True
c) i) Refer to Subsection 1.2.1
ii) Refer to Subsection 1.2.1
iii) Refer to Subsection 1.2.1
d) Refer to Fig. 1.4
2. a) i) uses a laser light as the source of illumination
ii) invented by Manfred von Ardenne
iii) uses electron as the source of illumination and 3-D image is
formed

iv) used for the preparation of ultrathin sections

v) useful for non-metallic and biological materials 37
Block 1 Techniques in Cell Biology
b) i) vacuum

ii) cooling

iii) tungsten

iv) Scanning Transmission Electron Microscopy (STEM)

v) Environmental Scanning Electron Microscope (ESEM)

3. a) i) interference

ii) compound light microscope

iii) fluorescein

iv) confocal microscopy

v) polarization microscopy

vi) formaldehyde and glutaraldehyde

vii) electron micrographs

viii) freeze-fracture

b) Refer to Subsection 1.4.2 and 1.4.3

Terminal Questions
1. a) Magnification is the measure of the ability of a lens or other optical
instruments to magnify. It is expressed as the ratio of the size of
the image to that of the object.

b) The smallest distance by which two points can be separated and

distinguished as individual objects is called resolution. It is
expressed in micrometres (µm).

c) Environmental scanning electron microscopy (ESEM) is a unique

system in which uncoated biological and industrial materials can
be examined with an electron beam in a high pressure
atmosphere. The specimens can be analyzed using ESEM without
destruction/distortion and additional specimen preparation
procedures. The surface of a solid sample is excited with a highly
focused energetic beam of electrons which induces X-ray
fluorescence from the elements within the sample.

d) Scanning transmission electron microscopy (STEM) combines the

principles of transmission electron microscopy and scanning
electron microscopy and can be performed on either type of
instrument. STEM requires very thin samples and looks primarily
at beam electrons transmitted by the sample. STEM is a
technically sophisticated microscope which is electronically
complex than a TEM or a SEM. The STEM technique scans a very
38 finely focused beam of electrons across the sample.
Unit 1 Microscopy
e) Negative staining method is the simpest technique used in TEM for
examining very small objects. Particles are suspended in a small
drop of liquid applied to copper grid and allowed to dry in air. After
drying, a drop of stain such as phosphotungstic acid or uranyl
acetate is applied to the surface.

f) Shadow-casting is a technique which shows the surface

texture of microscopic material. Sections or smears may be
studied throughout the whole range of microscopic magnification.
The method involves vacuum deposition of a metallic film on dried
specimens. Metal is deposited from an oblique angle so that it
coats some surfaces of specimen more than others.

2. The resolution is defined as the shortest distance between two points on

a specimen that can still be distinguished by the observer or camera
system as separate entities. The resolving power of a compound
microscope depends on (a) wavelength of light used to illuminate the
object, (b) refractive index of the medium between the object and the
objective, and (c) the semi vertical angle of the cone of light from the
object to the objective. The primary factor in determining resolution is the
objective numerical aperture.

3. Refer to Section 1.3.

4. a) A microscope is a combination of magnifying glasses. If one lens is

used in the microscope, it is called as simple microscope but when
multiple lenses are used, they are called compound microscopes.

b) Scanning Electron Microscope (SEM) uses electrons as the source

of illumination. The image is seen in 3-D. with high magnification
and high resolution. The specimen is coated with gold and the
electrons are reflected and give the details of surface topography
of the specimen.

Transmission Electron Microscope (TEM) also uses electrons as

the source of illumination and gives a 2-D view. Thin sections of
the specimen are obtained. The electron beams pass through the
sections and form an image with high magnification and high
resolution.

5. Refer to Subsections 1.2.4 and 1.2.5.

Acknowledgement
Fig 1.1 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2F50
a8d2.medialib.edu.glogster.com

Fig. 1.2 : Source:

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fmi
cro.magnet.fsu.edu%2Fprimer%2Fimages%2Fancient%2Fhoo
kemicro.jpg
39
Block 1 Techniques in Cell Biology
Fig. 1.3 : a) Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2
Fmedia.nationalgeographic.org%2Fassets%2Fphotos

b) https://www.google.co.in/imgres?imgurl=https%3A%2F%2
Fthumbs-prod.si-cdn.com)

Fig. 1.4 : Source: OpenStax Biology. Modification of work by

"GcG"/Wikimedia Commons
https://www.amscope.com/microscope-parts-and-functions

Fig. 1.5 : Source:

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fcd
n.britannica.com%2F50%2F114750-050-
06EEB5F0%2Fcompound-microscope.jpg

Fig. 1.12 : Source:

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fsta
tic.educalingo.com%2Fimg%2Fen%2F800%2Fmicrotome.jpg&
imgrefurl

Fig. 1.13 : Source:

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fim
age.slidesharecdn.com%2Fcellwall-151016090937

Fig. 1.16 : Source:

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fi.pi
nimg.com%2Foriginals%2F88%2F12%2Fe6%2F8812e60c6fa
054e8743ccb0f54d1ba8f.jpg&imgrefurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fww
w.pinterest.com%2Fpin%

Fig.1.19 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fars
.els-cdn.com%2Fcontent%2Fimage%2F1-s2.0-
S1369702110701437-
gr9.jpg&imgrefurl=https%3A%2F%2Ffanyv88.com%3A443%2Fhttps%2Fwww.sciencedirect.com

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