2 Polarizing Micros

POLARIZING MICROSCOPY
CONTENTS
INTRODUCTION
BIREFRIENGENT PRISMS
POLARIZING MICROSCOPE
MICROSCOPIC CONFIGURATION
PRINCIPLE
APPLICATIONS
REFERENCES
INTRODUCTION
POLARIZED MICROSCOPY is a Specialized Microscopy Technique
that elicits the optical properties of anisotropic materials revealing  structure and
composition. The polarized light microscope is designed to observe and
photograph specimens that are visible primarily due to their optically anisotropic
character.
Polarized light microscopes have a high degree of sensitivity and can be
utilized for both quantitative and qualitative studies targeted at a wide range of
anisotropic specimens, which is primarily employed in crystallography, represent a
far more difficult subject that is usually restricted to geologists, mineralogists, and
chemists. However, steady advances made over the past few years have enabled
biologists to study the birefringent character of many anisotropic sub-cellular
structures.
Polarized light
Natural sunlight and most forms of artificial illumination transmit light
waves whose electric field vectors vibrate in all perpendicular planes with respect
to the direction of propagation.
F C
H
J K
I
E G
Fig: Representation of vibration of light in all planes
When the electric field vectors are restricted to a single plane by filtration
then the light is said to be polarized with respect to the direction of propagation
and all waves vibrate in the same plane.
Such substances or anisotropic crystals capable of producing plane-
polarized light are called Birefringent.
Birefringent materials
Usually, when light passes through a medium such as glass (isotropic
materials), it is retarded and or refracted.
Dense medium Speed
(Glass)
Where as,
CRYSTAL Splits into two

Light ray
Anisotropic crystals such as quartz, calcite, and tourmaline and collagen

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 to an 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 velocities. This phenomenon
is termed double- or bi-refraction or Birefringence and is seen to a greater or
lesser degree in all anisotropic crystals.
Several prism based polarizing devices are available such as :
Nicol’s prism
Introduced by William Nicol in the year 1829
 He Cleaved and cemented calcite / Iceland spar with candabalsam
Diagram: Light ray (A) PRISM  (B) Polarized (C) Reflected out
If another prism placed above the first one:
Ray (B) – If optical path aligned – Pass through (B1)
Optical paths crossed  Reflected out (B2)
*Crossed when Upper prism rotated through 90°.
Polaroid discs (1935):
- glass or celluloid discs.
- Absorb the ordinary ray (which would be refracted out) only extraordinary
ray being transmitted.
- Ultramicroscopic crystals suspended in nitrocellulose.
- Mounted between two glass plates or celluloid sheet – act as single crystal.
Polarizing microscope involves interference of split light rays, as they are

reunited along the optical path.
Microscopic configuration:
- Contains unique additional components:

o Polarizer and analyzer.
o Compensation / retardation plates.
o Stain free objectives and condenser.
Principle:
Birefringent crystal if placed on stage of microscope having a polarizer in the sub

stage and an analyzer above the object plane:
Fig: A birefriengent crystal between crossed Nichol prisms
 With crossed polarizer and analyzer, and corresponding vibration directions of

crystal and polarizer – the crystal will appear dark on a dark background (A).
 If vibration directions do not correspond to the vibration of the polarizer, the two
rays transmitted by the crystal  will be resolved by analyzer – and the crystal
will appear bright on a dark background.
 The appearance of crystal is caused by the interference of two rays

recombined in the analyzer and is influenced by –
o Phase difference between two rays, which in turn depends on the

o Refractive indices of the crystal and its
o Thickness
If there is a Phase difference  Destructive interference occurs for some

wavelengths.
Other wavelengths reinforce   phase difference.
Series of interference colors
Orders (Each end with red)
I) Order – Black  Grey, white, yellow, orange, red.
II) Order Proceeds from red to blue, green, yellow, orange and finally red – Most
Brilliant.
Two phenomena are detected using polarizing light –
 Birefriengence.
 Dichroism.
Birefringence and Dichroism are optical properties of anisotrophic substances,

that is, substances that have different physical properties in different directions.
They are found for instance in crystals and are widely used in mineralogy as
qualitative tests for identification of various minerals.
Dichroism
The phenomena of producing plane polarized light by selective absorption

or change in intensity of light is dichroism. Either light of certain wavelength is
selectively absorbed, resulting in a characteristic color along a particular plane, or
a change in intensity of white light occurs when light passes through the substance
in particular planes. The dichroism shown by amyloid is a property of the
arrangement of the dye molecule – congo red or toluidene blue, being bound to the
protein in a highly oriented fashion so, producing different light absorbing
characteristics along certain planes of the fibril. Dichroism is studied in tissue
section by using polarized light with out an above stage analyzer filter.
Birefringence:
When light enters to an anisotropic crystal. It is refracted into two rays each
polarized with the vibrations directions oriented at right angles to one another and
traveling at different velocities.
Types of Birefringence:
1) Intrinsic or crystalline birefringence:
Anisotropy due to asymmetrical arrangement of clinical bonds, ions

or molecules
Common in collagen, muscle fibers, chromosomes

2) Form birefringence:
Found in mixed bodies where in asymmetrical particles of one RI are

dispersed in a specially oriented manner in a medium of high RI
Eg: filaments, sheets.
3) Strain Birefringence:
Distorted bonds due to mechanical stress  Birefringence
Glass, Elastic fibers under stress.
4) Positive and negative birefringence:
In a collagen fiber –
Slow ray (higher RI)  parallel to long axis of fiber  +ve birefringence.
Slow ray – perpendicular to long axis  -ve birefringence w.r.t. long axis.
Compensatory plates
Compensatory plate (quartz, selenite) – necessary to assess positive and negative

birefringence.
The compensatory plate Retards light (1/4)  interference pattern with red
background.
 Positive birefringent material – Blue
 Negative birefringent material – Yellow
*When its axis – secondary to the long axis of compensator.
Compensator plates can be made by – Apply two layers of clear adhesive tape to a
glass slide.
Applications / Uses of polarizing microscopy:
Identification of:
1. Exogenous crystalline material – Talk, silica.

2. Endogenous crystalline material – urate, Ca pyrophosphate.
3. Artifacts – Formalin pigment, starch  Poor fixation artifact; appear
 Stippled black (light microscopy)
 Bright white birefringence with polarized light.
4. Amyloid – Characteristic bright apple green birefringence with Congo red/

Sirius red.
5. Collagen (Picro Sirius / Van Gerion)
 Normal collagen
o Thin fibres – Green to greenish yellow.
o Thick fibres – Yellowish orange – orange red.
o Pathologic conditions (e.g. Fibroma) – Green – Greenish yellow.
6. Bone
 Woven bone (cross hetched) – Greenish yellow- Yellow.
 Lamellar bone (parallel) – yellow – orange red.
7. Dental tissues:
 Differentiate
o mantle dentine (orange)
o circumpulpal dentine (blue).
 Fiber orientation in
o Acellular cementum (fibres perpendicular to root surface) —

Orange
o Cellular cementum — Blue

REFERENCES
 Theory & practice of Histological techniques

 John D. Bancroft
 Cellular pathology technique
 CFA Culling
 Color Atlas and text book of Oral Anatomy, Histology and Embryology
 Berkovitz
 Clinicopathologic study of Ossifying Fibromas using van Gesion stain and
polarizing microscopy
 M.D.S. Dissertation - Dr. Santosh H.
 Central Odontogenic fibroma and hyperplastic dental follicle; Study with
picrosirius and polarizing microscopy JOPM 1996; 25; 125.
 www.microscope.org

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POLARIZING MICROSCOPY

Fig: Representation of vibration of light in all planes

Dense medium Speed

CRYSTAL Splits into two

Anisotropic crystals such as quartz, calcite, and tourmaline and collagen

Introduced by William Nicol in the year 1829

 He Cleaved and cemented calcite / Iceland spar with candabalsam

If another prism placed above the first one:

Ray (B) – If optical path aligned – Pass through (B1)

Optical paths crossed  Reflected out (B2)

*Crossed when Upper prism rotated through 90°.

Polaroid discs (1935):

- glass or celluloid discs.

- Ultramicroscopic crystals suspended in nitrocellulose.

Polarizing microscope involves interference of split light rays, as they are

- Contains unique additional components:

o Compensation / retardation plates.

o Stain free objectives and condenser.

Birefringent crystal if placed on stage of microscope having a polarizer in the sub

Fig: A birefriengent crystal between crossed Nichol prisms

 With crossed polarizer and analyzer, and corresponding vibration directions of

 The appearance of crystal is caused by the interference of two rays

o Phase difference between two rays, which in turn depends on the

If there is a Phase difference  Destructive interference occurs for some

Other wavelengths reinforce   phase difference.

Series of interference colors

Orders (Each end with red)

I) Order – Black  Grey, white, yellow, orange, red.

Birefringence and Dichroism are optical properties of anisotrophic substances,

The phenomena of producing plane polarized light by selective absorption

1) Intrinsic or crystalline birefringence:

Anisotropy due to asymmetrical arrangement of clinical bonds, ions

Common in collagen, muscle fibers, chromosomes

Found in mixed bodies where in asymmetrical particles of one RI are

Eg: filaments, sheets.

Distorted bonds due to mechanical stress  Birefringence

Glass, Elastic fibers under stress.

4) Positive and negative birefringence:

Compensatory plate (quartz, selenite) – necessary to assess positive and negative

 Positive birefringent material – Blue

 Negative birefringent material – Yellow

*When its axis – secondary to the long axis of compensator.

Applications / Uses of polarizing microscopy:

1. Exogenous crystalline material – Talk, silica.

3. Artifacts – Formalin pigment, starch  Poor fixation artifact; appear

 Stippled black (light microscopy)

 Bright white birefringence with polarized light.

4. Amyloid – Characteristic bright apple green birefringence with Congo red/

5. Collagen (Picro Sirius / Van Gerion)

o Thin fibres – Green to greenish yellow.

o Thick fibres – Yellowish orange – orange red.

o Pathologic conditions (e.g. Fibroma) – Green – Greenish yellow.

 Woven bone (cross hetched) – Greenish yellow- Yellow.

 Lamellar bone (parallel) – yellow – orange red.

o mantle dentine (orange)

o circumpulpal dentine (blue).

o Acellular cementum (fibres perpendicular to root surface) —

o Cellular cementum — Blue

 Theory & practice of Histological techniques