Phase Contrast Microscopy,by Dr Iram

POLARIZING MICROSCOPY, PHASE CONTRAST MICROSCOPY

Dr Iram Iqbal 1

Contents: II.Polarizing Microscopy

V.Phase contrast Microscopy

2

POLARIZING MICROSCOPY

3

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°.

4

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

5

6

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

7

8

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

9

10

TYPES OF POLARIZED LIGHT

11

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 rightelliptically 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.

15

17

18

19

20

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. 21

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. 22

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).

23

24

25

26

27

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.

28

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

29

METHODS OF POLARIZING LIGHT

30

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

31

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°.

32

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 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 sky and  the colours observed at sunset. sunset 35

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. 36 pyroxenes

37

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

38

 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

39

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 40

• 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

43

POLARIZING MICROSCOPY

44

PHASE CONTRAST MICROSCOPY

45

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. 46

BEHAVIOUR OF LIGHT

o o

When light from the microscope lamp passes through condensor and the specimen it can be divided into: Direct (undeviated light). light) Deviated (diffracted light). light) 47

•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 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

51

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. 52

53

 The lens of the eye-piece further magnifies this image wich is finally projected on the retina of the eye.

54

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. 55

 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.

56

57

58

59

 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.

60

61

62

63

64

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 65

66

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).

67

68

69

HALOS IN PHASE CONTRAST MICROSCOPY

70

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

71

72

73

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. 74

75

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 76

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 77

• c