microscope

1. -DR. PARIKSHYA SHRESTHA
-P.G. 1ST YEAR
-DEPT OF ORAL PATHOLOGY AND MICROBIOLOGY
-RAJARAJESWARI DENTAL COLLEGE AND HOSPITAL
MICROSCOPE - I

2. CONTENTS
 INTRODUCTION
 HISTORY
 TYPES OF MICROSCOPE
 COMPONENTS OF MICROSCOPE
 LIGHT AND ITS PROPERTIES
 LENS
 COMPONENTS OF MICROSCOPE IN DETAIL
 MAGNIFICATION AND ILLUMINATION
 MICROMETRY
 CLEANING AND MAINTAINANCE OF MICROSCOPE
 DARK GROUND MICROSCOPY
 POLARIZING MICROSCOPY
 PHASE CONTRAST MICROSCOPY
 INTERFERENCE MICROSCOPY
 DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY
 REFERENCE

3. Introduction
 The word microscope is derived from two Greek words ‘mikro’
meaning ‘small’ and ‘scopien’ meaning ‘to view’.
 Thus, it is an instrument which enables us to view small objects
by magnifying it and making it possible to be seen by the viewer.
 The science of investing small objects using such an instrument
is called Microscopy

4. History of microscope
1590 – Hans and Zachcharias Janssen of Holland claimed to
have invented microscope
1609 – Galileo Galileli developed a compound microscope with
convex, concave lens
1572-1633 – compound microscope with two convex lens, invented
in Rome by Carnelius Drebbel
1625 – Giovanni Faber coined the term microscope

5. History of microscope
1674 – Anton van Leeuwenhoek made and used a simple
microscope to view biological specimens.

6. Types of microscopes
Basically two types of microscope :
1. Simple Microscope : A simple microscope consists of a
single lens or a magnifying glass.
2. Compound microscope : A compound microscope consists
of two or more lenses.
Compound microscope can be further divided according to
different microscopic methods used .

7. Types of microscopes
Simple microscope Compound microscope

8. Microscopic Methods
A. Light Microscopy
1. Brightfield (light) microscopy
2. Darkfield microscopy
3. Phase-contrast microscopy
4. Interference microscopy
5. Differential interference contrast microscopy
6. Fluorescent microscopy
7. Polarizing microscope
B. Electron Microscopy
1. Transmission electron microscopy
2. Scanning electron microscopy
3. Scanning transmission electron microscopy
4. Atomic force microscopy
C. Scanning Probe Microscopes

9. Components of microscope

10. LIGHT
 Visible light is that portion of electromagnetic spectrum
that can be detected by human eyes, having wavelength
ranging from approx 400 nm to 800nm
 Light travels in straight lines. Light travels at different
speeds in air and in glass. Its path can be deflected or
reflected by means of mirrors or right angle prisms.

11. LIGHT
RETARDATION AND REFRACTION
 If light enters sheet of glass at right angle, light is retarded in
speed but direction is unchanged.
 If light enters the glass at any other angle, a deviation of direction
will occur in addition to retardation and this is called refraction.

12. LIGHT
 Light is slowed or retarded and “bent” or refracted
when it passes through air and enters a convex lens, gets
refracted when it leaves the convex lens and reenters air.

13. LIGHT
The curved lens will exhibit both retardation and refraction,
the extent of which is governed by :
a) Angle of incidence : angle at which light strikes the lens
b) Refractive index
c) Curvature of the lens
Angle of refraction : the angle to which the rays are deviated
within the glass or other transparent medium

14. LIGHT
 Refraction depends on the optical density of medium from
which lens is made, which is indicated by refractive index
(RI)
RI = velocity of light in air/velocity in subatance
 Refractive index is of great value in the computaion and
design of lenses, microscope slides and coverslip and
mounting media.
 RI of air = 1.00, water = 1.30, glass = 1.50

15. LIGHT
 Total internal reflection : light passing from glass into air
emerges parallel to the surface of lens if the angle of
incidence is increased. If the angle is too great, (critical
angle), the rays do not emerge but are totally internally
reflected.

16. LENS
 LENS
 Piece of glass or other transparent material, usually circular,
having the two surfaces ground and polished in a specific form
in order that rays of light passing through it shall either
converge or diverge.
 Two types of lens:
 Positive lens
 Negative lens

17. LENS
 Positive lens
 Thicker at centre
 Causes light rays to concentrate or converge to form a real
image
 Negative lens
 Thinner at centre
 Light passing will diverge or scatter and real image are
not seen

18. LENS
Focus
 When the lens concentrates the light rays to form a clear sharp
image of an object, the object is said to be in focus.
 The term ‘focus’ or ‘principal focus’ are used to indicate the
position in which a lens will form a sharp, clear picture of a
distant object.
 Conjugate foci : in addition to principal focus, a lens also has
conjugate foci; these are two points one on each side of the lens,
in one of which a clear image will be formed on a screen of an
object placed in the other.

19. LENS
 Image formation

20. LENS
Image formed
1. Real Image
 Formed by Objective lens of microscope
 Image is inverted, at greater magnification
 Can be seen on a screen

21. LENS
2. Virtual image
 Image formed within principal focus on same side of object
 Image appears the right way up and enlarged
 Cannot be focused on screen
 Formed by eyepiece of the microscope of real image
projected from the objective

23. Defects of Lens
1. Chromatic aberration
 Colours of white light, each refracted to different degree
 Shorter wavelength, greater degree of refraction
 White light entering a lens, on emerging forms a different
point of focus for each component colours, blue being
focused at a point nearer the lens than red
 Results in unsharp images with colored fringes

24. Defects of Lens
Correction : It is known as achromatism
 Done by using two component lens
 Positive lens is combined with a negative lens
 construction of compound lens of different glass element
a) Achromatic lens : will correct a thin positive lens for any two
colour
b) Apochromatic lens : fluorspar is incorporated, three colors can
be brought to the one focal point and the amount of chromatic
aberration visible in image is negligible.

25. Defects of Lens
2. Spherical aberration
 Due to entry of light rays into curved lens at its periphery
 Refracted more that at a centre and thus not brought to a
common focus
 Blurred image is formed
 Correction : same pattern used for chromatic aberration ie
using a powerful positive lens and partially neutralizing its
magnifying power with a negative lens made of glass having a
greater relative aberration

26. COMPONENTS OF MICROSCOPE

27. Eyepiece
 Function - to magnify the image formed by the objective
within the body tube, and present the eye with a virtual
image.
 Limits the field of view as seen by the eye
 Usually 5x, 7x, 10x, 20x, 40x type magnification are used.
 Can be used to correct the residual errors in objective lens.
 Undercorrected : blue ray refracted to greater degree than red;
blue fringe
 Overcorrected : orange fringe seen at edge of field diaphragm

28. Eyepiece
Types of eyepiece
1. Positive eyepiece : Ramsden eyepiece
2. Negative eyepiece : Huygenian eyepiece

29. Eyepiece
Positive eyepiece :
 With this, the focus is outside the
eyepiece lens system
 Field stop is outsidethe eyepiece,
from which the virtual image is
focused and magnified by entire
eyepiece.
 Ramsden eyepiece : lower lens has
its plane side towards object. These
are preferred for micrometer
eyepiece as they impart less
distortion to scales

30. Eyepiece
Negative eyepiece :
 With this, the focus is within the
eyepiece lens system
 Lower or field lens collects the image
that would have been formed by the
objective and cones it down to a
slightly smaller image at the level of
the field stop within eyepiece; upper
lens then produces an enlarged
virtual image that is seen by eye.
 Huygenian eyepiece : these are
undercorrected and are best suited
for use with achromatic objectives.

31. Eyepiece
 Other types :
 Compensating eyepiece
 Pointer eyepiece
 Micrometer eyepiece
 Double demonstration eyepiece
 High focal point eyepiece

32. Objective
 These are screwed into lower end of body tube by means of
standard thread, thus are interchangable.
 Designated by their focal length, dependent on tube length
 Consists of lenses and elements 5-15 in number, depending
on ratio, type and quality

33. Objective
 The main task of objective is to collect maximum light
possible from the object, unite it and form a high quality
magnified image some distance above.
 Every objective has a fixed working distance, focal length,
magnification and numerical aperture (NA) .

34. Objectives
Specifications mentioned on objective

35. Objective
 The working distance is the distance between an object in focus
and the front of the lens system of the objective.
 The focal length in the compound lens it is the distance between
an object in focus and a point approximately halfway between the
component lenses of the objective.
 Total Magnification is product of magnification values of
eyepiece and objective in a standard microscope.

36. Objective
Color Codes
 Microscope manufacturers label their objectives with color codes
to help in rapid identification of the objective.
Table : Color codes used for objectives
Magnification Color Code
4× Red
10× Yellow
40× Light Blue
100× White

37. Objective
Coverglass thickness
 is important if high-power 'dry’ objectives are being used, when
No. 1 coverglasses should be used, or an objective with a
correction collar may be employed which allows a range of
thickness or coverslip from 0.12 to 0.22 mm to be used (usually
0.17mm)
 oil-immersion objectives do not have coverglass restrictions since
they will have the same refractive index as the immersion oil.

38. Objective
Resolution
 The ability of the lens to distinguish fine structural details in a
specimen is known as the ‘resolving power’. It is the smallest
distance between two dots or lines that can be seen as separate
entities.
 It depends on the wavelength of light (λ) and the Numerical
Aperture of the lens.
 It is calculated as:
Resolution = 1.2 λ/2NA

39. Objective
Numerical Aperture
 This ability is of an object to resolve detail is expressed in terms
of numerical aperture(NA)
 Numerical aperture depends primarily on the extreme range of
the divergent rays that can be made to enter the lens (angular
aperture) and secondarily on the refractive index of the medium
between the object and the objective.
 The relation between numerical aperture, angular aperture and
refractive index is:
NA = n× Sin u
n=refractive index of medium between lens and object
sin u = the sine half the angle of aperture

40. Objective
Effect of high NA
 whilst a high numerical aperture increases the resolution
of an objective, it has the following disadvantages:
a) it reduces the depth of focus, i.e. the ability to focus on more
than one layer of an object at the same time, and
b) it reduces the flatness or the field, so that the edges are out of
focus.

41. Objective
Types of Objectives
 In most modern microscopes objectives are usually made up
more than one lens.
 This series of lenses is used to overcome certain limitations in
the lenses.

42. Types of Objective
 Achromatic: Corrected for two colors red and blue. It is the most
widely used for routine purposes.
 Fluorite: Corrected for yellow green color. Green light is
brought to a shorter focus and violet light to a longer focus.
 Apochromat: All colors are brought to same focus. It is fully
corrected for three colors. More highly corrected, often
incorporating fluorite glass. These lenses are used especially for
photomicrography and for screening cytological smears.
 Plan-achromat: Although histological sections are flat the
image produced by the microscope is not flat. It is saucer
shaped; it is not possible to focus the whole of the field sharply
at any one time. This aberration is corrected using flat-field
objective, also called as plan-achromat lenses.

43. Objective
Types of Objectives
Design and arrangement of lens in objective

44. Body tube
 Attached to limb, standard 160mm length
 Nosepiece/carrier for objectives fitted at lower end,
designated by number of objectives (2/3/4 based on
magnification needed.
 Three main forms : monocular, binocular and combined
photo-binocular

45. Body tube
Binocular
Microscope
Binocular tubes have provision made for
the adjustment of the interpupillary
distance, enabling each observer to adjust
for the individual facial proportion

46. Adjustments
a) Coarse adjustment knob
 enables the stage and substage to be moved rapidly up and
down
b) Fine adjustment knob
 enables the stage and substage to be moved slowly and
accurately
 Works by micrometer screws, levers and cams

47. Object Stage
A rigid platform above the condenser which supports the
glass slide is object stage.

48. Object Stage
 It has an aperture in the
center through which the light
can pass to illuminate the
specimen on the glass slide.
 The stage holds the slide
firmly and allows the slide
movements with a mechanical
vertical and horizontal
adjustment screws.

49. Object stage
 The mechanical stage is graduated with Vernier scales and the x
and y movements assist the operator to return to an exact
desired location in the specimen.
 Traveling range in most of the microscopes is 76 mm(X) 30 mm
(Y).

50. Illuminating apparatus
Substage :
 below the stage, attached to it and adjustable
 consists of:
1. Condenser
2. Iris-diaphragm
3. Filter carrier
4. Mirror

51. Condenser
 Light from the lamp is
directed into the sub stage
condenser either directly or
from a mirror or prism.
 The main purpose of the
condenser is to focus or
concentrate the available
light into the plane of the
object

52. Condensers
 Condensers should have the same numerical aperture as
objective.
 The ideal condenser should form a perfect image of the light
source.
 Three types of condensers are used :
 Abbe Condenser
 Aplanatic Condensers
 Achromatic Condensers

53. Iris diaphragm
 Also called aperture diaphragm
 Used to control the cone of light entering the condenser
 Intensity should always be reduced by using filters and not
by closing the diaphragm
 Adjustment of this iris diaphragm will alter the size and
volume of the cone of light focused on the object.

54. Iris diaphragm
 If the diaphragm is closed
too much, the image
becomes too contrasty and
refractile, whereas if the
diaphragm is left wide
open, the image will suffer
from glare due to
extraneous light
interference.

55. Iris diaphragm
 In both cases the resolution of the image is poor.
 The correct setting for the diaphragm is when the numerical
aperture of the condenser is matched to the numerical
aperture of the objective in use.

56. Filter carrier
 Usually a metal ring, pivoting on a screw to facilitate
the easy removal of filters

57. Mirror
 Plano concave mirror, fitted about 4 inch below stage
 Concave side have focus from object
 Plane mirror must always be used with condenser
 Built-in light source have mirrors fixed at the base.

58. MAGNIFICATION AND
ILLUMINATION

59. Magnification
 Magnification of lens will depend on its conjugate foci, i.e. the
distance from object to the lens and that from lens to image.
 Magnification is the product of the magnification of the
objectives and eyepieces and is dependent on following factors:
1. Focal length of objective
2. Distance between focal plane of objective and image it produces
3. Magnification of eyepiece
Magnification =Tube length × Eyepiece magnification
Focal length of the objective

60. Illumination
 Artificial illumination supplied by an electric filament lamp is
most commonly employed.
 Source of illumination should be:
1. Uniformly intense
2. Should completely flood the back lens of the condenser with
light when the lamp iris diaphragm is open
3. Make the object appear as though it were self-luminous
 In light microscope two different types of illuminations are used.
 • Critical illumination
 • Kohler illumination

61. Setting up microscope
Nelsonian method
 Light source should be
homogeneous and no lamp
condenser
 employed with bare light
source
 Light source should be
focused on the object plane
by racking the substage
condenser up or down

62. Setting up microscope
Kohler illumination
 Non homogeneous light
source
 Lamp condenser is essential
to project an image of lamp
filament onto the substage
iris diaphragm
 Lamp condensing lens
functions as light source
 Used with compound
microscope

63. Micrometry
 The standard unit of measurement in microscopy is a
micrometer(μm), which is 0.001mm
 To measure microscopic objects an eyepiece micrometer scale is
used in conjugation with stage micrometer.
A. Eyepiece micrometer
 Usually a disc on which arbitary scale is engraved
 Placed inside Huygenuan eyepiece, resting on the field stop.
 Gives sharp image of scale and have a greater eye clearance
B. Stage micrometer
 Consists of a 3 x 1 inch slide on which a millimeter scale is
engraved in 1/10 and 1/100 graduations

64. Cleaning and Maintainance
A. Daily cleaning routine
 Should be dusted daily and outer surface of lens of objective
polished with lens tissue or cotton wool
 Top lens of eyepiece polished to remove dust or fingerprints
and microscope set up for correct illumination. If dust still
present, eyepiece may need to be dismantled and both lenses
cleared.
 Substage condenser and mirror are cleaned in a similar
manner
 Removal of chemically active and sharp pieces of grits and
foreign material if present

65. Cleaning and Maintainance
B. Weekly cleaning routine
 Slides of adjustment, stage, substage wiped with cloth (in
xylene damp)
 Lens system checked
 Chip blower can be used for cleaning eyepiece and objective

66. Handling the Microscope
 Carry it with 2 hands-one on the arm and the other under the
base.
 Use lens paper (ONLY) to remove any oil from the 100X lens.
 Once oil has been added to the slide, do not move back to the
40X lens to focus: oil should never get on this lens. If this
happens, it will be very difficult to get all of the oil off.
 Turn the coarse adjustment knob so that the stage is far from the
lens.
 Place the microscope back into the correct spot in cabinet, with
the arm toward us, making sure that the 10X low power lens is
in place, pointing towards the stage-not the 100X oil immersion
lens. The lens could hit against the stage and get scratched.

67. DARK GROUND MICROSCOPY

68. Dark Field
 Fine structures can often not be seen in front of a bright
background as visibility is dependent on contrast between
object and the background.

69. Dark ground microscopy:
 the oblique light is thrown upon the object which does not
enter through the objective, they appear as self illuminous
objects on dark background.
 Only the reflected or scattered light forms the image of the
object.

70. Objectives and condensers:
 Objectives must have a lower
numerical aperture than the
condenser
 Low power- black
paper/glass inserted into filter
carrier.
 central rays are cut off and
peripheral rays from the
condenser passes through the
object but do not enter the
objective; the only light
entering the objective will be
that scattered by the object

71. Objectives and condensers:
 High power-special condenser
 Oil immersion must be used between the objective and object to
ensure maximum reflected light from the object enters the
objective
 Fixed-focus condenser: commonly used- thin glass slides and
coverslips( ideal no:1)

72. Setting up the microscope:
 Thin preparation- thin slide- coverslip
 Adjust light direct/through the condenser
 Place a drop of oil immersion on lower side of slide and also on top
lens of the condenser
 Move the rack up untill both surfaces meet without forming air
bubbles
 If correctly focused a small point of light will illuminate the object
on a dark background(low power)
 High power: place drop of oil on the coverslip and focused

73. Advantages and disadvantages
Advantages:
 Finer structures can be seen clearly hence can be best used
for spirochetes.
Disadvantages:
 Misleading impression of size
 When stained , difficult to see
 Need thinner sections without any refractory material like
oil, water droplets, air bubbles etc..

74. POLARIZING MICROSCOPY

75. POLARIZING MICROSCOPY
PRINCIPLE:
 Light rays when passed through a crystal are retarded in speed.
Being unevenly dense, the crystal will retard the rays to a different
degree hence the rays will be refracted or bent to differing degrees.
 This is known as DOUBLE REFRACTION OR BIREFRINGENCE
 The direction of vibration of the emergent rays will be at right
angles to each other.

76. Principle:
 A ray of light entering such a crystal will be converted
into two rays which will emerge at two different points.
 The emergent light rays will be polarized(one ray-on
single direction, second ray- single direction and right
angle to the first ray)

77. ISOTROPIC: substances through which light can pass in any
direction and at the same velocity , not able to produce polarized
light.
DICHROISM: A phenomenon given rise to by some substances and
crystals which can produce plane polarized light by differential
absorption.
PLEOCHORIC FILMS: Dichroic crystals are suspended in thin plastic
films and oriented in one direction .They can absorb all the colors
equally.

78. NICOL PRISM
 It is composed of a crystal made of Icelandic spar slit in half
and the halves cemented together with Canada balsam.
 Light rays having passed through it would emerge vibrating
in a single plane.
 The single direction in which the light is vibrating when it
emerges is known as the optical path of the prism.

79. POLAROID DISCS
 They are glass or celluloid covered discs with the ability to
polarize light
 Act as a single crystal of herpathite embedded in nitro cellulose
and mounted between plastic sheets which is not only
birefringent but has the ability to absorb the ordinary ray which
would be refracted out of a Nicol prism
 Only allows the extraordinary light to be transmitted.

80. MAIN COMPONENTS OF THE MICROSCOPE
The two polarizer's used are:
 POLARIZER
 ANALYSER

81. Polarization
Analyzer
Polarizer

82. POLARIZER
 Placed beneath the sub stage condenser
 Held in a rotatable graduated mount
 Can be removed from the light path when not required

83. ANALYSER
 Placed between the objective and eyepiece
 Is graduated-markings
 Enhances the image

84. POLARIZING MICROSCOPY
 In a polarizing microscope, a polarizing filter is placed
between light source and specimen.
 The second polarizer called analyzer is placed above
specimen between objective and eyepiece.

85. POLARIZING MICROSCOPY
 If the vibration directions
of the object correspond to
the vibration of the
polarizer when the
polarizer and analyzer are
at right angles there is
absence of light through
eyepiece.

86. Appearance of object depends on interference of the two
rays recombined in the analyzer which depends on phase of
difference between the two rays which in turn depends on the
difference in the two refractive indices of the crystal and on its
thickness
If the vibration direction do not correspond then the rays of
light transmitted by the object will be resolved in analyzer and
object appears bright on a dark background.

87. WORKING:

88. TYPES OF BIREFRINGENCE
 INTRINSIC OR CRYSTALLINE
 FORM
 STRAIN
 POSITIVE
 NEGATIVE
 QUARTZ AND COLLAGEN-POSITIVE BIREFRINGENCE
 POLAROID-
DISCS,CALCITE,URATES,CHROMOSOMES-NEGATIVE
BIREFRINGENCE

89. SIGN OF BIREFRINGENCE
 The ray passing through a medium of high RI is called slow and if
it passes through a medium of low RI it is called fast.
 If the slow ray is parallel to the length of the crystal, or fiber
birefringence is positive.
 If the slow ray is perpendicular to the long axis of the structure,
birefringence is negative.
 Determined by the use of a compensator either above the
specimen or below the polarizer at 45° to the direction of
polarized light.

90.  The compensator or specimen is rotated till the slow direction of
the compensator is parallel to the long axis of the crystal or fiber.
 The field is now red and if the crystal is blue the birefringence is
positive
 If the slow direction of compensator is parallel to the fast
direction of the crystal, it appears yellow and has negative
birefringence.

91. APPLICATIONS
It is used in the detection and observation of:
 Artifacts like formalin pigment
 Crystals of urate ,pyrophosphates etc
 Lipids, myelin etc
 Bone structure
 Proteins like collagen,amyloid,keratin
 Charcot-Leyden crystals, muscle striations etc.

92. PHASE CONTRAST MICROSCOPY

93. PHASE CONTRAST MICROSCOPY
 It is a technique which enables us to see very transparent
objects, which are almost invisible by ordinary transmitted light,
in clear detail and in good contrast to their surroundings, and to
see very small differences in thickness and density within the
objects.
 This is accomplished by converting these slight differences in
refractive index and thickness into changes of amplitude.

94. PRINCIPLE
 A ray of light is made of waves travelling together in a straight
line. When two such waves travel together,they are said to be in
phase. Such a ray will appear bright to the observer
 If one of the waves is held up or made to change the path, they
will no longer travel together and are said to interfere with each
other, differing in their intensity

95.  A special condenser and objective control the illumination in a
way that accentuates the differences in densities.
 It causes light to travel different routes through the various
parts of the cell
 The result is an image with differing degrees of darkness and
brightness collectively called contrast

96. Interference
CONSTRUCTIVE INTERFERENCE
 Light rays are in phase
 Amplitude or brightness is ‘doubled’ when recombined
DESTRUCTIVE INTERFERENCE
 Light rays are incoherent
 ½λ out of phase
 No light is seen
 Maximum interference

98. PARTS OF A PHASE CONTRAST
MICROSCOPE
ANNULUS:
 Made of opaque glass
 Has a hollow clear ring
 Can be centered by means of centering screws
PHAZE PLATE OR Z PLATE
 Clear glass disc with a circular trough etched in it to half the depth of disc
 The light passing through the trough has a phase difference of 1/4ƛ
compared to the rest of the plate
 Also contains a neutral density light absorbing material to reduce
brightness of direct rays.

99. PARTS OF A PHASE CONTRAST
MICROSCOPE
HIGH INTENSITY COMPOUND LAMP
 Usually used with a mercury green filter
AUXILLARY TELESCOPE
 Used in place of an eyepiece for examining the back focal
plane of the objective

100. Image formation in phase contrast microscope

101. WORKING OF THE MICROSCOPE
 Annulus is placed in the condenser and the phase plate is placed
in the objective
 It allows only a small ring of light to pass into the microscope
 The phase plate has a circle engraved on it which should match,
with the ring of light coming in from the annulus through the
condenser.

102.  Some rays of light will pass through unaltered while some
rays will be retarded or diffracted by approximately 1/4ƛ.
 On passing through a phase plate the diffracted ray is
retarded further by 1/4ƛ and will now interfere with the
direct light ray.
 The total retardation of diffracted rays is now 1/2ƛ and
interfere will produce image contrast thus revealing even
small details.

103. APPLICATIONS
 For examining unstained bacteria
 For examining wet preparations of specimens
 For examining faecal preparations for trophozoites or amoebae
 In searching for trypanosomes in blood and other body fluids

105. INTERFERENCE MICROSCOPY
 generates mutually interfering beams which produce the
contrast. It is this feature which enables very small phase
changes to be seen and measured.
 The two rays which eventually combine to produce image are
formed by a plate of birefringent material placed immediately
above the condenser.
 These two rays having passed through the object plane are
recombined by a similar plate of birefringent material below the
front lens of the objective.

106.  One ray passes through a point
in the object and the other
through an area adjacent to it.
 Each point in the final image is a
compound one made up of two
mutually interfering rays.
 A special Wollaston prism is
added to the condenser to split
the beam of light and also to
recombine the two dissimilar
beams.

107. APPLICATIONS
 To study individual parts of living cells with maximum
resolution of detail
 To estimate dry mass when it is applied as a highly accurate
optical balance
 To assess section thickness of specimen.

108. DIFFERENTIAL INTERFERENCE
CONTRAST MICROSCOPY
 Designed by Nomarski hence also called as Nomarskis
microscope.
 Relies on the interference of a pair of wave fronts to
generate contrast.

109. It comprises of:
-a polarizer
-a condenser with a modified Wollaston prism
-a beam splitting slide
This slide consists of a modified Wollaston prism oriented at
45° to anattached analyzer,mounted in an adjustable carriage
and accommodated in the analyzer slot between the
objective and the eyepiece.

110. Working of the microscope
 Polarized light passes through the prism below the
condenser
 The prism below the condenser acts as a compensator
 Every interference fringe of the upper prism is correlated
with an interference fringe of the same order but opposite
sign in this compensator

111.  The two rays pass in turn through the condenser the
object and the objective before passing through the
second prism and analyzer.
 The upper prism can be moved laterally enabling the rays
to be displaced laterally or sheared before being
recombined in the analyzer when they undergo
interference.
 This produces ‘interference contrast’ and together with
rotation of the polarizers enhances the three-dimensional
(3D) effect in the image.

113. ADVANTAGES
 Wide variety of interference colors can be used
 Improved image contrast
 No phase halo included
 Lateral shearing of rays is reduced so that excellent three
dimensional images can be produced.

114. USES
 As an infinitely variable phase contrast microscope,
individual parts of living cells can be studied.
 As a highly accurate optical balance, used for estimating dry
mass down to 1x10 gm
 Quantitative measurement of phase change or optical
path difference
 Studying live and unstained biological samples such as a
smear from a tissue culture or individual water borne celled
organism

117. References
 Textbook of oral pathology – Jaypee brothers, 1E; Anil Ghom,
Shubhangi Mhaske
 Histology A Text and Atlas - With Correlated Cell and Molecular
Biology, 7E (2015) ; Wojciech Pawlina
 Bancroft’s Theory and Practice of Histological Techniques ,7th edition
 CFA Culling’s Histological techniques
 Essentials of Microbiology; Surinder Kumar ,1st edition, 2016
 MICROSCOPE Basics and Beyond, Revised edition 2003, Mortimer
Abramowitz