POLARIZING

MICROSCOPY,
PHASE CONTRAST
MICROSCOPY

Dr Iram Iqbal

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Contents:

II.Polarizing Microscopy

V.Phase contrast Microscopy

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POLARIZING
MICROSCOPY

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 LIGHT WAVE
 Light can be thought of as a wave,
wave that
vibrates back and forth as it moves.
 Individual light waves each have their
own wavelength,
wavelength as well as direction
of vibration.
vibration
 Angles of vibration can range through
a full 360°.

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 UNPOLARIZED LIGHT
WAVE
 The electric and magnetic vibrations of an
electromagnetic wave occur in numerous
planes.
 A light wave which is vibrating in more than
one plane is referred to as unpolarized
light.
light

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POLARIZATION OF LIGHT
 The process of transforming unpolarized light
into polarized light is known as polarization.
polarization

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 POLARIZED LIGHT WAVES
Polarized
light waves
are light
waves in
which the
vibrations
occur in a
single
plane.
plane
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TYPES OF POLARIZED
LIGHT

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 Classification of Polarization
Linear Polarization
A plane electromagnetic wave is said to be linearly polarized.
The transverse electric field wave is accompanied by a
magnetic field wave as illustrated.
 Circular Polarization
• If light is composed of two plane waves of
equal amplitude but differing in phase by
90°, then the light is said to be circularly
polarized.
• If you could see the tip of the electric field
vector, it would appear to be moving in a
circle as it approached you. If while
looking at the source, the electric vector of
the light coming toward you appears to be
rotating counterclockwise, the light is said
to be right-circularly polarized.
• If clockwise, then left-circularly
polarized light.
• Another way of saying it is that if the
thumb of your right hand were pointing in
the direction of propagation of the light,
the electric vector would be rotating in the
direction of your fingers.
• Circularly polarized light may be produced
by passing linearly polarized light
through a quarter-wave plate at an angle
of 45° to the optic axis of the plate.
 Elliptical Polarization
• Elliptically polarized light
consists of two
perpendicular waves of
unequal amplitude which
differ in phase by 90°. The
illustration shows right-
elliptically polarized light.
•
• If the thumb of your right
hand were pointing in the
direction of propagation of
the light, the electric vector
would be rotating in the
direction of your fingers.
• Compare with linear and circular polarization
 POLARIZING FILTERS
 A Polaroid filter is able to polarize light
because of the chemical composition of the
filter material. The filter can be thought of as
having long-chain molecules that are
aligned within the filter in a verticle or
horizontal direction.
 Since unpolarized light is made up of both
vertical and horizontal ways the filter only
lets about half of he waves through..
 When unpolarized light is transmitted
through a polarizing filter,it emerges with
one half intensity and with vibration in a
single plane.
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 CROSSED POLARS
When two polarizing
filters are placed
such that the
vibrational directions
of light passed
through each are at
right angles;
angles then no
light will pass
through.
This is referred to as
crossed polars.
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 PARALLEL OR OBLIQUE
POLARS
If the second
polarizing filter is at
any other orientation;
orientation
some light will pass
through.
The amount depends
upon whether the
filters are closer to
being parallel or
crossed.
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BIREFRINGENCE

 Birefringence, or double refraction,

refraction is the
decomposition of a ray of light into two rays
(the ordinary ray n0 and extraordinary ray
ne),
ne when it passes through certain types of
material, such as calcite crystals or boron
nitride, depending on the polarization of the
light.
 This effect can occur only if the structure of
the material is anisotropic (directionally
dependant).
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 ISOTROPICS
 Isotropics are the substances through which
light can pass in any direction and at the
same velocity and are not able to produce
polarized light.
 Non crystalline materials , glasses and
some plastics are isotropics.

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 ANISOTROPICS
 Anisotropics are the
materials in which the
optical properties vary
with crystal orientation
because the spacing of
atoms in crystal
structures differ along
the three axis.
axis
 e.g; Collegen,
cellulose, cholesterol
crystals, microtubules,
microfilaments.
microfilaments
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METHODS OF POLARIZING
LIGHT

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 POLARIZATION BY
TRANSMISSION
 A Polaroid serves as a device which filters out
one-half of the vibrations upon transmission
of the light through the filter.
filter

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 POLARIZATION BY
REFLECTION
 Unpolarized light strikes a smooth surface, such as a
plane of glass, tabletop, and asphalt roadways the
reflected light is polarized such that its vibration
direction is parallel to the reflecting surface.The
reflected light is completely polarized only when the
angle between the reflected and the refracted ray =
90°.

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 POLARIZATION BY
REFRACTION,
• Refraction occurs when a
beam of light passes from one
material into another material.
• At the surface of two materials
the path of the beam changes
its direction.
• The refracted beam acquires
some degree of polarization.
• Most often the polarization
occurs in a plane perpendicular
to the surface.
 POLARIZATION BY
REFRACTION
 This method of
producing plane
polarized light was
employed prior to
selective absorption
in microscopes.
microscopes
 The most common
method used was
the Nicol Prism.
Prism
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 POLARIZATION BY
SCATTERING
 The absorption and remission of
light waves causes the light to be
scattered about the medium. this
scattered light is partially polarized
 This is observed as light passes
through our atmosphere. the
scattered light often produces a
glare in the skies.
 blue colour of the sky and
 the colours observed at sunset.
sunset

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 POLARIZATION BY
SELECTIVE ABSORPTION
 This method is used to produce plane polarized
light in microscopes,
microscopes using polarized filters.
 Some anisotropic materials have the
ability to strongly absorb light vibrating in
one direction and transmitting light vibrating
at right angles more easily.
easily
 The ability to selectively transmit and absorb
light is termed ple och ro ism,
ism seen in
minerals such as tourmaline, biotite,
hornblende, (most amphiboles), some
pyroxenes.
pyroxenes 36
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 USES OF POLARIZATION
 Polaroid sunglasses
 Photography
 Nicol prism
 Liquid crystal
display (LCD) used
in numerous
applications including
wrist watches,
computer screens,
timers, clocks, and
many others.
others
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 Light reflected by the flat surface of a
highway is partially polarized with the electric
field vectors vibrating in a direction that is
parallel to the ground. This light can be blocked
by polarizing filters oriented in a vertical
direction with a pair of polarized sunglasses.
sunglasses

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POLARIZING MICROSCOPE
 The polarizing microscope is
particularly useful in the study of
birefringent materials such as
crystals and strained non-crystalline
substances.

 It is widely used for chemical

microscopy and optical mineralogy.
mineralogy

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• When a birefringent substance is
rotated between two polarizers`which
are crossed the image appears and
disappears alternately at each 45
degree of rotation. in a complete
revolution of 360 the image appear 4
times and 4 times it disappears
completely
• When one of the planes of the vibration of
the object is in a parallel plane to the
polarizer only one part of the ray can
develop, and its further passage is blocked
by the analyzer in the crossed position.
• At however phase difference between the 2
rays which can develop are able to combine
in the analyzer and form a visible image
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POLARIZING
MICROSCOPY

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PHASE CONTRAST
MICROSCOPY

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 PHASE CONTRAST
MICROSCOPY
 Was first described by
Dutch Physicist Frits
Zernike.
Zernike
 It is a contrast
enhancing optical
technique that can be
used to produce high
contrast images of
transparent
specimens which are
unstained and alive.
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 BEHAVIOUR OF LIGHT
When light from the
microscope lamp
passes through
condensor and the
specimen it can be
divided into:
o Direct (undeviated
light).
light)
o Deviated (diffracted
light).
light)
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 •Illumination bypasses
Specimen > no phase shift

•Illumination passes
through thin part of •Illumination passes
Specimen > small phase through thick part of
retardation Specimen > larger phase
retardation

In Phase Contrast, the Intensity of contrast is dependent on

- the differences of Refractive Indices of the Specimen and the
surrounding Medium, and
- The Thickness and Native Contrast of the Specimen.
Results:
Improved contrast
differences
between the
specimen and the
surrounding
medium making it
possible to see
cells and cell
organelles
without staining.
 OPTICAL PRINCIPLES
 Constructive interference: Two rays of
same frequency when combined will
double in amplitude or brightness if they
are in phase with each other.
 Destructive interference: If the rays are
out of phase with each other there is
decrease in the resultant amplitude.
Difference in amplitude while maintaining
maximum interference occurs in phase
contrast microscopy. 50
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 PRINCIPLE OF PHASE
CONTRAST MICROSCOPY:
Phase contrast microscopy imparts
contrast to unstained biological
material by transforming phase
differences of light caused by
differences in refractive index
between cellular components into
differences in amplitude of light,
light i.e.,
light and dark areas, which can be
observed.
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 The lens of the eye-piece
further magnifies this image
wich is finally projected on the
retina of the eye.

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Phase contrast objects alter the light
diffracted by the specimen by retarding
such light by approximately ¼ of a
wavelength by the refractive index (n),
compared to the undeviated light passing
through or around the object unaffected.

Our eyes are unable to detect these

phase differences. The human eye is
only sensitive to colours of visible
spectrum, which is variations of light
frequency, and levels of light intensity.
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 If the refractive index of the specimen
is greater than that of the surrounding
medium, the wave is reduced in velocity
while passing through the specimen and
is subsequently retarded in relative
phase when it emerges from the
specimen.
 If the refractive index of the
surrounding medium exceeds that of
the specimen, the wave is advanced in
phase upon exiting the specimen.

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 The microscope condenser images the
annular diaphragm at infinity.
infinity

 The objective produces an image at the

back focal plane (where a conjugate
phase plate is positioned).

 A phase plate is mounted in or near

the objective back focal plane in order
to selectively alter the phase and
amplitude of the surround light passing
through the specimen.
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 PHASE PLATES
Rings in phase plate
can include
– Attenuating layer
(absorption but
no phase shift), or
– Phase-shifting
layer (no
absorption, phase
shift only),
only or
– Any combination
of the two
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 POSITIVE AND NEGATIVE
PHASE CONTRAST
 Positive Phase
Specimen appears
dark against light
background (usual
now).

 Negative Phase
Specimen appears
bright against dark
background (looks
like darkfield optics).
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 HALOS IN PHASE
CONTRAST MICROSCOPY

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 Reducing Phase Halo

 Modification of specimen and medium.

 Use of non-osmotic solutes to

increase medium index of refraction.

 Use of apodized phase plates.

plates

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 THINGS OBSERVED IN
PHASE CONTRAST
MICROSCOPY
Phase contrast objects (Unstained
specimens that do not absorb light):
 Living cells (usually culture)
 Microorganisms
 Thin tissue slices
 Subcellular particles
 Cell nuclei and organelles.
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 APPLICATIONS OF PHASE
CONTRAST MICROSCOPY
 Widely applied in biological and medical
research, especially throughout the fields of
cytology and histology.
histology
 Employed in diagnosis of tumor cells and the
growth, dynamics, and behavior of a wide
variety of living cells in culture.
 Other areas in the biological arena that benefit
from phase contrast observation are hematology,
virology, bacteriology, parasitology,
paleontology, and marine biology.
 Industrial and chemical applications for phase
contrast include mineralogy, crystallography,
and polymer morphology investigations.
investigations
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REFERENCES:
 Theory and Practice of Histological
Techniques by John D.Bencroft 5th edition
 An Introduction to Histotechnology by
Geoffrey G. Brown
 Text book of Histology by Leeson, Leeson,
Paparo 5th edition
 Phase Contrast Microscope -
Information.htm
 Wikipedia free encyclopedia
 Google search for images for phase contrast
and polarizing microscopy

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