phase contrast microscopy,by dr iram

Dr Iram Iqbal

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POLARIZING MICROSCOPY,PHASE CONTRAST MICROSCOPY

Contents:

I.Polarizing Microscopy

II.Phase contrast Microscopy

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POLARIZING MICROSCOPY

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LIGHT WAVE Light can be thought of as a wavewave, that

vibrates back and forth as it moves. Individual light waves each have their

own wavelengthwavelength, as well as direction direction of vibrationof vibration.

Angles of vibration can range through a full 360°.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 unpolarized lightlight.

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POLARIZATION OF LIGHT

The process of transforming unpolarized light into polarized light is known as polarizationpolarization.

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POLARIZED LIGHT WAVES

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Polarized light waves are light waves in which the vibrations occur in a single single planeplane.

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TYPES OF POLARIZED LIGHT

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Classification of Polarization

Linear PolarizationA 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.

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• 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 moleculeslong-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

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When two polarizing filters are placed such that the vibrational directions of light passed through each are at at right anglesright angles; then no light will pass through. This is referred to as crossed polars.

PARALLEL OR OBLIQUE POLARS

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If the second polarizing filter is at any other orientationany other orientation; some light will pass through.The amount depends upon whether the filters are closer to being parallel or crossed.

BIREFRINGENCE

Birefringence, or double refractionBirefringence, or double refraction, is the decomposition of a ray of light into two rays (the ordinary ray n0 and extraordinary ray ordinary ray n0 and extraordinary ray nene), 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 anisotropic (directionally dependant)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 , glassesNon crystalline materials , glasses and and some plasticssome plastics are isotropics.

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ANISOTROPICS Anisotropics are the

materials in which the optical propertiesoptical properties vary with crystal orientation because the spacing of spacing of atomsatoms in crystal structures differ along the three axisthree axis.

e.g; Collegen, Collegen, cellulose, cholesterol cellulose, cholesterol crystals, microtubules, crystals, microtubules, microfilamentsmicrofilaments.

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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 filterlight through the filter.

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POLARIZATION BY REFLECTION

Unpolarized light strikes a smooth surface, such as a plane of glass, tabletopplane 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 = angle between the reflected and the refracted ray = 90°.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 microscopesin microscopes.

The most common

method used was

the Nicol PrismNicol Prism.34

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 skyblue colour of the sky and

the colours observed at sunsetthe colours observed at sunset.

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POLARIZATION BY SELECTIVE ABSORPTION

This method is used to produce plane polarized light in microscopesmicroscopes, using polarized filters.

Some anisotropic materialsSome anisotropic materials have the ability to strongly absorb light vibrating in strongly absorb light vibrating in one direction and transmitting light vibrating one direction and transmitting light vibrating at right angles more easilyat right angles more easily.

The ability to selectively transmit and absorb light is termed ple och ro ismple och ro ism, seen in minerals such as tourmaline, biotite, minerals such as tourmaline, biotite, hornblende, (most amphiboles), some hornblende, (most amphiboles), some pyroxenespyroxenes. 36

USES OF POLARIZATION

Polaroid sunglasses Photography Nicol prism Liquid crystal Liquid crystal

display (LCD)display (LCD) used in numerous applications including wrist watches, wrist watches, computer screens, computer screens, timers, clocks, and timers, clocks, and many othersmany others.

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Light reflected by the flat surface of a reflected by the flat surface of a highwayhighway 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 sunglassespair of polarized sunglasses.

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POLARIZING MICROSCOPE

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

It is widely used for chemical chemical microscopymicroscopy and optical mineralogyoptical 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 Dutch Physicist Frits ZernikeZernike.

It is a a contrast contrast enhancing optical enhancing optical techniquetechnique that can be used to produce high contrast images of contrast images of transparent transparent specimensspecimens 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 DirectDirect (undeviated (undeviated light)light).

o DeviatedDeviated (diffracted (diffracted light)light).

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•Illumination bypasses Specimen > no phase shift

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

•Illumination passes through thick part of 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 frequencysame frequency when combined will double in amplitude or brightness if they are in phasein phase with each other.

Destructive interference: If the rays are out of phaseout 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

PRINCIPLE OF PHASE CONTRAST MICROSCOPY:

Phase contrast microscopy imparts imparts contrast to unstained biologicalcontrast to unstained biological material material by transforming phase by transforming phase differences of light caused bydifferences of light caused by differences in refractive indexdifferences in refractive index between cellular components intobetween cellular components into differences in amplitude of lightdifferences in amplitude of 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 velocityreduced in velocity while passing through the specimen and is subsequently retarded in relative retarded in relative phasephase when it emerges from the specimen.

If the refractive index of the surrounding medium exceeds that of the specimen, the wave is advanced in advanced in phasephase upon exiting the specimen.

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POSITIVE AND NEGATIVE PHASE CONTRAST

Positive Phase Positive Phase SpecimenSpecimen appears dark against light dark against light backgroundbackground (usual now).

Negative Phase Negative Phase SpecimenSpecimen appears appears bright against dark bright against dark backgroundbackground (looks like darkfield optics).

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THINGS OBSERVED IN PHASE CONTRAST

MICROSCOPY Phase contrast objects (Unstained Unstained

specimensspecimens 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 histologycytology and histology.

Employed in diagnosis of tumor cells and the growth, dynamics, and behaviorgrowth, dynamics, and behavior of a wide variety of living cells in culture.

Other areas in the biological arenaOther 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, mineralogy, crystallography, and polymer morphology investigationsand polymer morphology 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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