Polarized microscopy seminar/prosthodontic courses

POLARIZED
MICROSCOPY
INDIAN DENTAL ACADEMY
Leader in
continuing Dental
Education
www.indiandentalacademy.com

REFERENCES
• Theory and Practice of Histological
Techniques-Bancroft
• Cellular Pathology Technique-Culling
• Microscope-Basics & Beyond
• J.K. Soneja, Microscopy

CONTENTS
• History & Introduction of polarized
microscopy
• Polarized light
• Birefringence
• Constituents of polarized microscope
• Dichroism
• Applications of polarized microscope
• Advantages & disadvantages of polarized
microscope
• Conclusion

ADVANCES IN MICROSCOPY
Over the last 150 years , there has been a
painstaking development of new techniques
for light and electron microscopy, which
facilitates the precise analysis of cell & tissue
structure and demonstrate more clearly the
changes , which pathological processes bring
about.

ADVANCES IN MICROSCOPY
• Dark field microscopy
• Fluorescence microscopy
• Phase contrast microscopy
• Polarized microscopy
• Electron microscopy
• Stereomicroscopy

POLARIZED MICROSCOPY

How to compare & contrast polarized
microscope and light microscope ??

LIGHT SOURCE
STAGE ANALYZER
How to compare & contrast polarized
microscope and light microscope ??

HISTORY
• Polarizing microscope was
developed in the late nineteenth
century.
• It was initially made for studying
rocks and minerals.
• It may also be used in the biology
and medical fields.
• Polarizing microscopes are also
known as petrographic
microscopes.

INTRODUCTION
• A polarizing microscope is a microscope that uses polarized
light for looking at specimens.
• It is used for detailed examination of specimens.
• Numerous crystals , fibrous structures, pigments , lipids ,
proteins, bone and amyloid deposits exhibit birefringence.

POLARIZED LIGHT
• Transverse wave light whose vibration possess direction is
called polarized light.
• Light from an ordinary light source (natural light) that
vibrates in random directions is called non-polarized light.
• Natural light vibrates in many planes or vibration directions
whereas polarized light vibrates in only one plane .

plane of
vibration
vibration
direction
propagation
direction
light vibrates in
all planes that contain
the light ray
(i.e., all planes
perpendicular to
the propagation
direction
POLARIZED LIGHT UNPOLARIZED LIGHT

POLARIZER
• Polarized light can be produced for
microscopy purpose by passing natural light
through a polarizer.
• Polarizer is an optical component made
from a substance that will allow vibrations of
only one vibration direction to pass.

Light passes through the polarizer
West
(left)
east
(right)
Plane polarized lightUnpolarized light

BIREFRINGENCE
• Birefringence or double refraction
is the phenomenon, which occurs
when light that is launched through a
crystal material is divided into two
linearly polarized light rays having
mutually crossing vibration
directions, and then refracted.

• Their velocities and refractive
indices(RI) differ from one another.
• Higher the RI greater the retardation of
the ray.
• Substances or crystals capable of
producing plane polarized light are
called birefringent .

mineral
grain
plane polarized
light
fast ray
(low RI )
slow ray
(high RI)
lower polarizer
D=retardation
d
Observation:
frequency of light remains
unchanged during splitting,
regardless of material
F= V/ l
if light speed changes, l must
also change
l is related to color; if l changes,
color also changes
Violet (400 nm)  Red (700 nm)
BIREFRINGENCE

BIREFRINGENCE
WAVELENGTH IN NANOMETRES
Thicknessinmicrons
T

NICOL PRISMS
• Named after the inventor .
• Polarizers made from calcite, were cemented together
with Canada balsam in such a way that slow ray was
reflected away from optical pathway , and into the
mount of prism , leaving only polarized fast ray to pass
through.

POLAROID DISCS
• In 1935, polaroids- glass or celluloid covered discs with the
ability to polarize light were made available for use in place of
Nicol prisms.
• Polaroids are made by suspending ultramicroscopic crystals
of herapathite in nitrocellulose.
• Some substances and crystals which can produce plane
polarized light by differential absorption give rise to the
phenomenon called dichroism.

• Such crystals suspended in thin plastic films in one
direction have now replaced the bulky and
expensive Nicol prisms.
• These thin films totally absorb the slow rays and are
pleochroic (absorbing all colors equally) .

POLARIZED LIGHT MICROSCOPE CONFIGURATION

CONSTITUENTS OF POLARIZED
MICROSCOPE
Polarizing microscope uses two polarizers-
1. Polarizer & Analyzer
Polarizer
• Placed beneath the substage condenser
• Held in rotatable graduated mount
• Can be removed from light path when not required

ANALYZER
• It is the upper polarizer
• Placed between the objective and the eyepiece
• Also graduated for measurement to be taken .

A microscope equipped for polarized light

2. Polarizing objective ( strain free objective)
• An objective lens for polarization is
designed and manufactured to have
low turbulence by refraction in the
polarizing state on the lens surface .
• Also, to have low lens strain .
• A polarizing objective can be
distinguished from the ordinary ones
by the label P, PO, Pol .

3. Polarizing condenser
A polarizing condenser has following 3
characteristics –
• Built-in rotatable polarizer
• Top lens-out construction when parallel
light illumination at low magnification is
required
• Strain-free optical system, like the
objectives

4. Polarizing rotating stage
• Rotating an anisotropy between crossed nicols changes the
brightness.
• For this reason, in polarized light observation, the specimen
is often rotated to the diagonal position (the position where
the anisotropy is brightest).

• In other words, rotatability of the polarizing stage
and centerability are fundamental .
• 360° angle gradations are indicated in the area
surrounding the rotating stage .

5. Bertrand lens
• A Bertrand lens projects an interference image of
the specimen, formed in the objective pupil, onto
the objective image position .
• It is located between the analyzer and eyepiece for
easy in and out of the light path.

6. Centerable revolving nosepiece
• Since the stage needs to be rotated for
polarizing observation, the objective and
the optical axis of the tube must coincide
exactly with one another.
• In order for the optical axis to completely
match even when the lens‘ magnification is
changed, a revolver with optical centering
mechanism is installed in each hole.

7. Eyepiece with crosshair
• The eyepiece has a built-in focusing plate containing a
crosshair.
• By inserting the point pin into the observation tube sleeve, the
vibration direction of the polarizer and analyzer can be made
to agree with the crosshair in the visual field.

8. Test plate with compensator
• These are phase plates used for verifying the double
refractivity of specimens,
• determining their vibration directions , and for retardation
measurement .

Polarizer and Analyzer are the essential
components of the polarizing microscope
When we look through 2 polarizers, there can be 3 conditions:-
1. When polarizer and analyzer are parallel
Rays vibrating in the parallel plane are able to pass.
-

2. When polarizer and analyzer are crossed
Rays able to pass the polarizer are blocked by analyzer.
The condition when no light reaches the observer is known
as Extinction .

west (left)
east (right)
south
(front)
north
(back)
Black!!
POLARIZER ANALYZER
The condition when no light reaches the
observer is known as EXTINCTION.

Now insert a thin section of a rock
west
(left)
east
(right)
Unpolarized light
Light vibrating
in many planes
and with many
wavelengths
Light and
colors reach
eye!
Light
vibrating
E-W

3. When a birefringent substance is rotated between
crossed polarizer
It is visible when it is in the diagonal position (i.e.,
when it is halfway (45º) between the vibration planes
of polarizer).

• At 45 degree, however phase differences between 2 rays
which can develop are able to combine in the analyzer and
form a visible image.
• Image appears and disappears alternately at each 45
degree of rotation.

Two Phenomenon in polarizing microscope
that are important for Histopathologists are :-
a) Birefringence - Property of resolving a beam
of plane polarized light into 2 rays at different
velocities.
b) Dichroism

Dichroism
• Some birefringent substances are dichroic.
• When a dichroic substance is rotated in polarized light
(i.e. using polarizer only).
• Changes of color and intensity can be seen after
rotating 90 degrees.
• The original color returns after a further 90 degree
rotation.
• This is due to the differential absorption of light.www.indiandentalacademy.com

DICHROISM

APPLICATIONS OF POLARIZING
MICROSCOPY IN HISTOPATHOLOGY
• Artifacts : Formalin pigment, sutures , starch
• Crystals : Talc , urate , pyrophosphate , silica ,
etc.
URIC ACID CRYSTALSwww.indiandentalacademy.com

• Protein : Collagen , amyloid , keratin
Glomerulus from renal biopsy stained with
congo red and examined by polarization
microscopy. The characteristic "apple-green"
birefringence of amyloid is apparent .

APPLICATIONS OF POLARIZING
MICROSCOPY IN HISTOPATHOLOGY
• Lipids : Myelin
• Bone structure : Osteoid seams , woven bone
• Miscellaneous : Muscle striations , charcot- layden
crystals
• On routine light microscopy, the distinction between
dentin and cementum is often difficult, but the use of
polarized light clearly separates the two different layers.

• Graphite implantation within oral mucosa appears
similar microscopically to amalgam but can be
differentiated by its pattern of birefringence after
treatment with ammonium sulfide and by the lack of
staining of the reticulin fibres.
• In OSMF, to analyze collagen distribution in different
stages using the picrosirius red stain under polarized
microscope.

In Dentigerous cysts, thin fibres
showed greenish yellow color and
thick fibres showed orange- red color.
• To compare the pattern of collagen fibres in odontogenic cysts .
Section of OKC showing
orangish red birefringence pattern
of collagen fibres

ADVANTAGES OF POLARIZING
MICROSCOPE
• They have a compact size, making them convenient for
transporting.
• A polarized microscope uses an optical device known as a
"polarizer “ which configures the movement of light and
forces its vibration in a single direction.

• Polarizing microscopes are often customized, designed and
constructed to suit the application which will be viewed.
• Polarized light generates clearer images and higher
magnification of the specimen than a general compound light
microscope.
• Moreover, polarizing microscopes are equipped with a rotating
stage which helps in generating an intensified image of the
entire specimen.

Disadvantages of polarized microscope
• Even using phase-polar illumination, not all the fibers present
may be seen.
• The method requires a great degree of sophistication on the
part of the microscopist.
• The method uses only a tiny amount of material for analysis.
This may lead to sampling bias and false results.

CONCLUSION
• Microscopes dedicated for use with polarized light are very
sophisticated instruments having components specifically
designed to minimize strain and provide sharp, crisp and clear
images of birefringent specimens.
• For simple qualitative work , a standard miroscope can be
converted for polarized light studies .

THANK
YOU !!
I
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Polarized microscopy seminar/prosthodontic courses

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