• Father of Biotechnology: Karl Ereky- (October 20, 1878 – June 17, 1952) was a Hungarian agricultural

engineer.

“Biotechnology is a branch of science that deals with biology based techniques

that harnesses cellular and biomolecular processes to develop technologies and
products that help improve our lives and the health of our planet”

• Biotechnology utilizes organism or part of organism or biological processes for the benefit of mankind.

e.g.
A) Making wine: (Whole organism)
Grape Juice + Yeast Alcohol + Carbon dioxide.

B) Hepatitis B Vaccine- (Part of the organism)

Also known as recombinant vaccine. It is the first anti cancer vaccine that prevents liver cancer.

C) Golden rice- (A process)

Yellow in colour. β- Carotene Vitamin A
 Microscopy is defined as lens or combination of lenses used to magnify and observe near objects so
the details that are invisible to our naked eye can be revealed.

o Microscopy serves two independent function:

(i) Magnification: enlargement of the object

(ii) Resolution: the shortest distance between two points on a

specimen that can still be distinguished by an observer.

Limit of Resolution Unresolved

 Resolution (d) =λ / 2 NA
where, λ= wavelength of the light
NA= numerical aperture

• The wavelength of light is inversely

proportional to the magnification of
microscope
Role of immersion oil in optical microscopy

• NA (numerical aperture) of a lens is based on the

refractive index of the immersion medium.

 i.e. NA= n x sin α

where, n = refractive index (n = 1 for air; n = 1.51 for oil)

α = angular aperture of the objective lens

 POINTS TO BE REMEMBER:

o Higher the resolving power of the lens, smaller the limit of resolution.

o Resolution of a light microscope depends on:

a) Wavelength of the light source

b) Angular aperture of the optical component

c) Refractive index of the medium surrounding the specimen

 TYPES of Microscopy:

o Depending on the lens system:

1) Simple microscope 2) Compound microscope

o Depending on the source of illumination:

1) Light microscope 2) Electron microscope

• Difference between light and electron microscope:

Light microscope Electron microscope

Employs optical lenses Employs electromagnetic

lenses
Light as source of radiation Electron beam as source of
radiation
Radiating light travels in air Emitting electrons travel in
medium vacuum
It has the resolution of 0.2 It has the resolution of 0.5
µm nm
N.B. Electrons have shorter wavelength than
Can magnify the object to Can magnify the object to visible light, and this allows electron
1000-1500 times over 100000 times microscopes to produce higher-resolution
images than standard light microscopes.
Parts of light microscope
Eyepiece: The lens the viewer looks through to see the specimen. The
eyepiece usually contains a 10X or 15X power lens.

Diopter Adjustment: Useful as a means to change focus on one eyepiece so

as to correct for any difference in vision between your two eyes.

Body tube (Head): The body tube connects the eyepiece to the objective
lenses.

Arm: The arm connects the body tube to the base of the microscope.

Coarse and Fine adjustment: Brings the specimen into general focus and fine
tuning to increase the detail of the specimen.

Nosepiece: A rotating extension that houses the objective lenses. Viewer can
spins the nosepiece to select different objective lenses.

Objective lens: Lenses closest to the specimen. A standard microscope has

three, four, or five objective lenses that range in power from 4X to 100X.

Stage: The flat platform where the slide is placed.

Stage clips: Metal clips that hold the slide in place.

Stage height adjustment (Stage Control): These knobs move the stage left
and right or up and down.

Aperture: The hole in the middle of the stage that allows light from the
illuminator to reach the specimen.

On/off switch: This switch on the base of the microscope turns the illuminator
off and on.
 1. Bright field microscopy

It is the standard microscope that is used in Cellular Biology, and

Microbiological Laboratory studies.
Principle: It is based on the
• For a specimen to be the focus and produce an image under the
bright field microscope, the specimen must pass through a uniform
beam of the illuminating light.
• Through differential absorption and differential refraction, the
microscope will produce a contrasting image.
• Can view: objects with natural pigmentation,
fixed and stained objects or
objects which are thick enough to absorb a significant
amount of light despite being colourless.
• Max. magnification is 1000x
• Resolution of such microscope is 0.00027 mm (0.27µm), i.e. 1/1000th
the thickness of a human hair.
• Bright field microscopy ray diagram
 2. Dark field microscopy

• Dark-field microscopy is ideally used to illuminate causing

them to appear brightly lit against a dark background.
• To view a specimen in dark field, an opaque disc is placed underneath the
condenser lens, so that only light that is scattered by objects on the slide can
reach the eye.
• The light at the apex of the cone is focused at the plane of the specimen; as
this light moves past the specimen plane it spreads again into a hollow
cone.
• The objective lens sits in the dark hollow of this cone; although the light
travels around and past the objective lens, no rays enter it.
• The entire field appears dark when there is no sample on the microscope
stage; thus the name dark-field microscopy.
• When a sample is on the stage, the light at the apex of the cone strikes it.
The rays scattered by the sample and captured in the objective lens thus
make the image.
• It is useful for finding cells in suspension and viewing living microbes.
 3. Phase contrast microscopy

• It was first described by Dutch physicist Frits Zernike.

• Principle: The basic principle lies of the fact that although biological structures are highly transparent to visible light,
they cause phase changes or retardations in transmitted radiation. This minute variations in phase is translated into
corresponding changes in amplitude, which can be visualized as differences in image contrast.
• It is a contrast enhancing optical technique that produce high contrast images of transparent, unstained
specimens that do not have sufficient contrast for bright field.
• Living cells (e.g. dynamic mobility of mitochondria), microorganisms, thin tissue slices, etc. can be seen/observed
under phase contrast microscope.
• Resolution = 0.1nm
 In brightfield, when imaging translucent samples, image contrast, ΔI, only indicates absorption of light by the sample.
 In phase contrast, the phase plate converts phase differences due to scattering in the sample into amplitude changes via
constructive and destructive interference between background and scattered light; this results in increased contrast between
the background and the sample.
 Phase contrast microscopy beam diagram.

 The condenser annulus allows only a hollow focused cone of light to illuminate the sample (light blue);
 After passing through the sample, scattered light (orange) is delayed by -¼λ (typical).
 When undeflected light - primarily background illumination - passes through the phase ring of the phase plate, it is
shifted +¼λ and dimmed 50% (dark blue).
 These phase shifts result in constructive and destructive interference between background and scattered light at the
image plane.
4. Atomic Force Microscopy (AFM) [a.k.a Scanning Force Microscope (SFM) or Scanning Probe Microscope (SPM)]

• Invented by Binning and colleagues in 1986.

• It was developed to overcome a basic drawback with STM (that can only image conducting or semiconducting
surfaces).
• The AFM is an influential surface analysis technique that enables the imaging at nanoscale resolution of almost
any type of surface, including - polymers, ceramics, composites, glass, and biological samples.
• Principle: AFM is based on the measurement and localization of different forces such as van der Waals
molecular forces, magnetic forces, contact forces, adhesion strength between a tip and the sample to measure
the sample topography or mechanical properties.
The quantity of the generated force between the tip and the surface depends on the spring constant (stiffness)
of the cantilever and the distance between the tip and the surface.

This force can be characterized with Hooke’s Law.

F = -kz (F = Force; k = spring constant;
z = the distance the lever is bent or
cantilever deflection)
Construction and Working:
 AFM consists of a spring like cantilever carried by a support system.
 The cantilever at one end is attached by a sharp tip which act as a
probe.
 A piezoelectric element oscillates the cantilever.
 An XYZ axis drive permits to displace the sample which is mounted on the
sample stage in X,Y,Z directions with respect to the tip apex.
 The AFM operates by measuring the force between a probe and the
sample.
 The cantilever with its sharp tip (probe) scanned the specimen surface.
 Height variations in the sample will change the deflection of the
cantilever.
 The detector records the deflection signal and motion of the cantilever.
 The deflections which are at the atomic scale phenomenon are
converted into an electrical signal, a macro scale phenomenon and
digitized to provide a three dimensional image of the surface.
Operation Modes of AFM:

Contact mode In contact mode, the tip is in a soft physical contact with the surface.
• The tip is able to move above the surface with a specific height or under a constant
force.
• The movement is strongly influenced by frictional and adhesive forces that can cause
damage to the sample.
• When the spring constant of cantilever is less than surface, the cantilever bends. The
force on the tip is repulsive.
• By maintaining a constant cantilever deflection (using the feedback loops) the force
between the probe and the sample remains constant and an image of the surface is
obtained.

Non contact mode In this mode tip does not touch the sample.
• The tip usually hovers about 5–15 nm and oscillates above the surface during scan.
• It uses feedback loop to monitor changes in the amplitude due to attractive van der
Waals forces so the surface topography can be monitored.
• It is better in soft samples.

Dynamic (Tapping) mode This mode eliminates the frictional force by intermittently
contacting the surface and oscillating with sufficient amplitude to prevent it from being
trapped in by adhesive forces.
• This mode of operation is less destructive than contact mode.
• The cantilever oscillates nearby its resonance frequency.
• An electronic feedback loop provides the oscillation amplitude remaining constant so
that a constant tip-sample interaction is conserved during the scan.
 Why silicon or silicon nitride is use in AFM?

LAO: Localized Anodic Oxidation is the

formation of an oxide layer on top of a
substrate by attracting OH- ions near the Si or
Si3N4
surface.
• The oxidation takes place when a negatively
polarized conductive tip is placed at close
range of a grounded sample.
• The high electric field created around the
sharp tip, causes the breakage of water
molecules near the interface into ions H+,
OH- and O-. The OH- and O- ions are then
pushed away from the biased tip and forced
into contact with the samples surface.
 Applications of AFM

 It is used for imaging, measuring matter at the

nanoscale.

 Visualization of single protein molecules at

submolecular resolution.

 Biological interactions such as

 antibody-antigen recognition,
 To study the mechanical behaviour of the
 protein-ligand binding,
cellular structure and its interaction with
 cDNA base pairing
extracellular matrix or cell-cell interaction.
 Bacterium-surface interaction
 5. INTERFERENCE MICROSCOPY

 It is an optical microscopy technique that uses interference between two white light illumination beams to generate an image
with enhanced contrast.

 Principle:
The interference microscope works on the principle that “although biological structures are highly transparent to
visible light, they cause phase changes or retardations in transmitted radiations”.

 It offers additional advantage over phase contrast microscope by giving quantitative data.
 It detects small and continuous changes in refractive index whereas phase microscope reveals only sharp boundaries.

Some example of interference that we see in daily life:

Interference Patterns occur in disc Soap Film Interference Patterns Interference Structures in Butterfly Wings
Instrumentation and Working:

• The laser (L) beam is divided on the beam splitter S.

• The reference beam reflects from the control mirror M2 and

the object beam transmits through the object and reflects

from the mirror M1.

• The reference beam and the beam transmitted through the

object and reflected interfere on the detector where the optic

path difference between the two beams is estimated.

• The variations in phase can be transformed in coloured

images that a living cell may resemble a stained preparation.

 6. POLARIZATION MICROSCOPY

• What is polarized light? • Light is an electromagnetic wave i.e. it has both electric and magnetic
waves vibrating at different planes.
• Polarized waves are light waves in which the direction of vibration is the
same for all waves, only one plane.
• The process of transforming unpolarized light into the polarized
light is known as polarization.

• When polarized light is propagated through a component material with

the same velocity, independent of the impinging direction, they are
called as isotropic.

• Anisotropic crystals and biological specimen have crystallographically

distinct axes and interact with light in a manner that is dependent upon
the orientation of the crystalline lattice with respect to the incident light.

• When light enters a non-equivalent axis in a anisotropic crystal, it is

refracted into two rays each polarized with the vibration directions
oriented at right angles to one another, and traveling at different
retarded velocities. This phenomenon is known as double- or bi-
refraction (or birefringence)
• The refracted light are seen to a greater or lesser degree in all
anisotropic crystals.
• Certain components of cells and tissues show particular behaviour when examined
under polarized light.
• Some structures affect the velocity of light producing birefringent (two different
indexes of refraction).
• Birefringence (B) forms the principle of a polarization microscopy.
• It is expressed quantitatively as the difference between the two indexes of refraction.
These differences in indexes result into retardation of polarized light.
• This retardation depends upon the thickness (t) of the specimen.
𝝀
Therefore, B = (Ne- No)=
𝒕

Working:
• The microscope consist of both a polarizer, positioned in the light path before the specimen, and
an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and
the observation tubes or camera port.
• When light passes through the first polarizing filter, linearly polarized light is produced. If the linearly
polarized light passes through a birefringent material in the correct polarization plane, it is refracted and
split into two sister rays, and the polarization plane of a portion of the rays is turned by 90°. The refracted
light rays then pass through the second polarizer (analyzer), if it is aligned correctly (i.e. 90° relative to the
first polarizing filter).
• After exiting the specimen, the light components become out of phase, however the analyzer recombined
the constructive and destructive interference when they pass through this second polarizer.
 Differential Interference Contrast Microscopy (DIC)

PRINCIPLE: it is based on interferometers where light from a single source is split into two beams which then combined to
produce interference.

Construction or Components of DIC:

1) Polarizer – This produces a single beam of plane polarized light from the lamp.
2) Modified Wollaston prism – This produces two beams of light, polarized at right angles to one another, slightly
out of phase with one another, and having a very small amount of shear. The amount of shear is on the order of the
resolution of the objective lens.
3) Condenser – The condenser focuses the two slightly sheared beams on the specimen plane.
4) Specimen – The specimen causes a change in optical path difference between the two beams. This change is
sensitive to very closely spaced variations in the specimen's refractive index and/or thickness since the beams
themselves are very closely spaced.
5) Objectives Lens – A DIC objective lens is placed between the sample stage and the second prism.
6) Second modified Wollaston prism – A second prism is placed above the objective lens. It emphasizes optical
path differences introduced by the specimen.
7) Analyzer – This is a second polarizer placed after the second modified Wollaston prism. This polarizer has its
major transmission axis at right angles to the lower polarizer.
8) Eyepiece – Interference of the two modified beams occurs in the intermediate image plane forming a real image
that is further magnified by the eyepiece.
 WORKING OF DIC:
• Unpolarised light entering the microscope
gets polarized at 45º by polarizing filter
and enters the first Wollaston prism.
• The first prism separates the incoming
light into two rays polarised at 90º to each
other, known as the sampling and
reference rays.
• Condenser lens focused the two rays and
allowed to pass through two adjacent
points in the sample, around 0.2 µm apart.

• Due to difference in refractive index or thickness of the sample the ray upon passing the sample will experienced a change in phase.
• Objective lens then focussed the ray for the second Wollaston prism.
• The second prism recombines the two rays into one polarised ray at 135º.
• This combination of ray (after the 2nd prism) leads to interference (brightening or darkening of the image), due to optical differentiation,
generating the image of the sample under observation.
• Uses: For imaging live and unstained biological samples.