Introduction History Compound microscope Variants of microscopes Dark field microscope Phase contrast microscope Fluorescent microscope Polarising microscope Electron microscope A Microscope is an instrument for viewing objects that are too small to be seen by the naked/ unaided eyes. In Greek micron= small skopien=to look at The science of investigating small object using such an instrument is called microscopy The term microscopic means minute or very small, not visible with the eye unless aided by a microscope From ancient times, man wanted to see things for smaller than could be perceived with the naked eye. This led to the construction in the 16th century, of a magnifier composed of a single convex lens, and this in turn led to the eventual development of the microscope. The most famous early pioneers in the history of microscope are Digges of England and Hans & Zcharias Janssen of Holland It was Antony Van Leeuwenhoek who became the man to make and use a real microscope. Leeuwenhoek microscope was called as single lens microscope because it had convex lens attached to metal holder and was focused using screws

1. MICROSCOPY

2. • Introduction
• History
• Compound microscope
• Variants of microscopes
– Dark field microscope
– Phase contrast microscope
– Fluorescent microscope
– Polarising microscope
– Electron microscope

3. INTRODUCTION
• A Microscope is an instrument for viewing objects that
are too small to be seen by the naked/ unaided eyes.
In Greek micron= small
skopien=to look at
• The science of investigating small object using such an
instrument is called microscopy
• The term microscopic means minute or very small, not
visible with the eye unless aided by a microscope

4. History
• From ancient times, man
wanted to see things for
smaller than could be
perceived with the naked
eye.

5. • This led to the construction in
the 16th
century, of a magnifier
composed of a single convex
lens, and this in turn led to the
eventual development of the
microscope.

6. • The most famous early
pioneers in the history
of microscope are
Digges of England and
Hans & Zcharias Janssen
of Holland

7. • It was Antony Van
Leeuwenhoek who
became the man to
make and use a real
microscope.
Leeuwenhoek ground and polished small
glass ball into lens with a magnification of
270x and used this lens to make the worlds
first practical microscope

8. • Leeuwenhoek microscope was
called as single lens
microscope because it had
convex lens attached to metal
holder and was focused using
screws

9. • The compound microscope system was invented in
the 17th
century.
• This type of microscope incorporate more the one
lens so the image magnified by one lens can be
further magnified by another lens.

10. • In 17th-century Robert
Hooke, using compound
microscope, discovered the
fact that living things were
composed of cells.

11. • Today, the term
"microscope" is
generally used to refer
to this type of
compound microscope

12. • In 19th century there was a
dramatic progress in the
development of the
microscope.
Carl Zeiss, devoted significant effort to the
manufacture of microscopes, Ernst Abbe,
carried out a theoretical study of optical
principles, and Otto Schott, conducted
research on optical glass.

13. • In 1931, Ruska & Knoll contributed to first
electron microscope.
• In 1940, Fluorescent Microscope developed.
• In 1953, Fredrick Zernike developed Phase
Contrast Microscope.

14. Types of microscope
• Simple microscope : one or several lenses
mounted closely together .e.g. simple hand
lens
• Compound microscope : widely separated
lenses fitted into a instrument.

15. Compound Microscope
Components: 4 Systems
- the support system
- the magnification system
- the illumination system
- the adjustment system

16. The Support System
Consists of
– the foot
– the limb
– the revolving nose piece
– the stage
– the mechanical stage
The foot and limb supports the microscope.
The body tube of the microscope is attached to
limb.

17. Revolving Nose Piece
(Objective Changer)
A Rotating device to which objectives are
attached
- Should move smoothly.
- Should have distinct click feel when an
objective is properly seated.

18. The Stage:
Rigid platform where objects are to be
examined.
- Has aperture of 1-1.5 inch
- Provided with metal clips, micrometer threads
- Movement occurs in 2 directions.
- Standard stage: 3x1 inch slide moves over an
area of 3.5x1.25 inch.

19. The magnification system
• Consists of system of lenses
• Lenses mounted in two groups- one at either
end of body tube
1. Objective lenses: located at bottom end of
tube- above the object.
2. Eye piece lenses: located at the top end of
tube.

20. Objectives:
– Interchangeable
– Magnifying power of each, shown by figure
engraved on sleeve of objectives
• 10X objective magnifies 10 times
• 40X objective magnifies 40 times
• 100X objective magnifies 100 times.
Magnification is defined as the degree of
enlargement of objective achieved by
microscope.

21. Types of objective:
• Achromatic
• Apochromatic
• Fluorite objectives/semi-
apochromatic
• Planachromat obj.: used for
photomicrography
• Polarizing obj.
• Phase obj.

23. Numerical Aperture (NA):
The NA is defined as the ratio of
the diameter of the lens to its
focal length
=indicates the amount of light
that enters an objective from
a point in the microscopic field.
NA= n X sinµ
n=RI of media air=1 oil=1.5
µ=angle at the aperture formed by two extreme rays
NA of dry lense= 0.65
NA of oil immersion= 1.28 or 1.30

24. Objectives may have equal focal length, but different
numerical apertures… depending on the diameter of front
lenses.
>NA: more the resolving power.
>NA: smaller the front lens of objective
Resolving Power (RP): of a microscope is its ability to reveal
closely adjacent structured details as separate or distinct.
RP of : Human Eye= 0.25 mm
: Compound Microscope= 0.25 µm
: Electron Microscope= 5Å or 5 nm

25. RP= 0.61x λ [λ= wave length]
NA [NA= numerical aperture]
RP depends upon:
1. Wavelength of light used
2. Numerical aperture
Shortest wavelength gives max. resolution
Immersion oil increases RP by conversing many light rays
that would be lost in dry objective by refraction.
>RP– clearer the image

26. Working distance (WD): of an objective is the
distance between the front lens of the objective
and object slide when the image is in focus.
Magnifying power increased as WD decreases
WD of X10 is 5-8 mm
X40 is 0.5-1.5 mm
X100 is 0.15-0.20 mm

27. Eye Piece:
The eye piece magnifies
the real image produced
by the objective.
Magnifying Power is marked on the eye piece
– X5 eye piece magnifies 5 times the image
produced by objective
– X10 eye piece magnifies 10 times.

28. Huygenian eyepiece:
Focus is b/w the lenses of
eye-piece, lower field lens
collects the image formed
by the objective ,cones it
down to a slightly smaller
image at the level of
diaphragm within the eye-
piece ;the upper lens then
produces an enlarged
virtual image.

29. • Ramsden eyepiece:
The focus is outside
the eye piece,
diaphragm is outside
the piece from which
the virtual image is
focused & magnified
by the entire eyepiece

30. Total magnification:
• Magnification occurs at two
level one at objective and
other at the eyepiece level.
• Total magnification=
power of eyepiece X power of
the objective.

31. • The magnification depends on-
-Mechanical tube length
-Focal length of the objective.
-The magnifying power of eye piece.
• Magnification
=tube length X mag of eye piece
Focal length of objective

32. The Illumination system
• Light source:An electric light is preferable. Can
be from infront or from self illumination
within the microscope.
• Mirror:
– Reflects rays from the light source on to the
object
– One side has a plane surface and other side a
concave surface.

33. Condenser:
• Brings the rays of light to a common focus on
the object to be examined.
• Between the mirror and stage.
• Max illumination=condenser raised
• Min illumination=condenser lowered
• Must be centered and adjusted correctly

34. Diaphragm:
• It is placed inside the condenser used to
reduce or increase the angle and therefore
also amount of light passes into the
condenser.
• Wider the diaphragm-more the numerical
aperture and smaller the detail seen.

35. Filters:
• A blue ray light filter is fitted undernaeth the
microscope condenser.
• Cuts out glare.
• Sharpens detail and is restful to the eyes.

36. The Adjustment System
-Course adjustment screw
-Fine adjustment screw
-Condenser adjustment screw
-Condenser centering screw
-Iris diaphragm lever
-Mechanical stage control

37. Coarse adjustment screw:
• Largest screw
• Used to achieve appropriate
focus.
Fine adjustment screw:
• Moves the objective slowly
• Used to bring the object into perfect focus
Condenser adjustment screw:
• Used to raise or lower the condenser :

38. Condenser centering screw:
• Three screws placed around the condenser.
• Used to centre the condenser.
Iris diaphragm lever:
• Small lever fixed to the condenser.
• It can be moved to close or open the diaphragm. Thus
increases or decreases both the angle and the intensity
of the light.

39. Mechanical stage control:
• Used to move the object slide on the stage.
• Two screws: one moves backward or forwards
and other moves left and right.

40. Setting up the microscope
1. Place the microscope on a firm bench with
a square felt pad under the microscope.
2. Turn on the light source.
3. Back off the coarse focus to raise the
nosepiece.
4. Place the specimen slide on the stage and
secure in the proper position, look at the
slide and place it, so that the specimen is
over the light aperture on the stage.

41. 6. Lower the objective lens close to the slide.
7. Centering the condenser:
- Lower the condenser, open the iris diaphragm.
- Exam with lowest power objective, focus the slide using coarse
adjustment.
- Close the diaphragm: A blurred circle of light with dark ring appears,
raise the condenser for sharp focus of ring.
- Adjust the mirror or self illuminating unit for bright ring.
- Adjust the centering screws of the condenser so that the circle of light
is in exact centre of the field.

42. 8. Focusing the objective
– Low Power: rack down the condenser to the bottom, lower
the objective until it is just above the slide. Raise the objective
until the clear image is seen.
– High Power 40x: Rack down the condenser half way down,
lower the objective until it is just above the slide.
– Oil immersion 100x: Dry stained preparation must be used.
Place a tiny drop of immersion oil on the part to be examined.
Rack the condenser up, open the iris diphragm fully. Bring the
objective close to the slide. Using the coarse and fine
adjustments screws, focus the image

43. Micrometry
• The standard unit of measurement in microscopy is
micrometer.
• An eye piece micrometer scale is used along with stage
micrometer to measure the microscopic objects.
• Eyepiece micrometer scale:graduated scale mounted on
the diaphragm.
• Stage micrometer :microscopic slide bearing an engraved
scale 1mm in length and graduated at 0.01mm[10µm]

44. Method:
1. Insert a eyepiece micrometer scale and place stage
micrometer on the stage.
2. Select the objective to be used when measuring the
object and focus on the stage micrometer scale.
3. Determine the number of divisions of the eye piece scale
equal to an exact number of divisions of the stage
micrometer scale
4. Remove the stage micrometer, focus on the object to be
measured and determine the number of eyepiece
division exactly covering the object

45. Calculate the size of the object as follows assuming 100
eye piece division is equal to 10 stage division and the
object was covered by 12 eye piece division-
100 eye piece division=10 stage division
1 stage division= 0.01[10µm]
Therefore,100 eye division=100µm
Therefore,1 eye piece division=1µm
Therefore,12 eye piece division=12µm
The diameter of object=12µm

46. Cleaning the microscope
Materials:
• Clean piece of old cloth,fine linen
handkerchief.
• Lens tissue paper,white absorbent paper.
• Soft camel hair brush or fine paint brush or
blower for cleaning lens.
• Cleaning solution containing 80% petroleum
ether and 20% 2-propanol.

47. Method:
• Cleaning the optical surface-
-Must be kept free from dust with a fine
paint brush
-Oil residues should be removed with
special lens tissue paper,absorbent
paper.
-Finally cleaned with a cleaning solution.

48. • Cleaning the instrument:
-Heavy contamination can be removed
with mild soapy solution.
-Grease and oil removed with cleaning
solution.
-Instrument cleaned with a 50:50
mixture of dist water and 95% ethanol.

49. Maintaining the microscope
• Check the mechanical stage
• Check the focusing mechanism.
• Remove any fungal growth.
• Check the diaphragm
• Clean all mechanical parts.
• Lubricate the microscope.
• Check the optical alignment.

50. Care of microscope
• Never carry the microscope limb with one
hand.
• Avoid touching the bulbs with your fingers.
• Never dip the objectives in xylene or ethanol.
• Never use ordinary paper to clean the lenses.

51. • Never clean the inside lenses of eyepiece and
objective with cloth/paper.
• Never leave the microscope without eyepiece.
• Never press the objective on to the slide.
• Never touch the with fingers.

52. BINOCULAR MICROSCOPE
• A microscope fitted with
double eyepieces for vision
with both eyes.
• Reduce eyestrain and
muscular fatigue which may
result from monocular, high-
power microscopy.

53. FOCUSSING BINOCULAR MICROSCOPE
1. Turn on the light source. Binocscopes have either a built
in unit or an external power supply.
2. Switch to the 10x objective lens.
3. Adjust the coarse focus to raise the nose piece (or lower
the stage).
4. Clip the specimen slide on the stage in the proper
position.

54. 5. Look at the ocular lenses of your scope. One lens is
fixed and the other has a focusing ring (like a pair of
binoculars). Bring the lens as close to the slide as
possible, then, looking only through the fixed ocular
lens, back off until the specimen just comes into focus.
Adjust fine focus similarly for the fixed lens.
6. Now, looking only through the adjustable ocular, adjust
its focus using the focus ring around the lens. Look with
both eyes (adjust for interpupillary distance to see a
single round lighted field) and make any minor
adjustments to focus.

55. 6. Center the image and adjust the light using the
condensor lens, iris diaphragm and light source .
7. Recenter and adjust focus, first coarse, then fine focus
as in 5.
8. Readjust diaphragm as needed.
9. Now switch objectives to a higher power. Readjust fine
focus and light (diaphragm)

56. DARKGROUND MICROSCOPY
• Optical microscopy illumination
technique used to enhance the contrast in
unstained transparent samples.
• Principle: The condenser directs the light
to hit the specimen at an oblique angle.
only light that hits objects in the specimen’
will be deflected upwards in to the objective
lens for visualisation.
• All other light that passes through the
specimen will miss the objective thus
making the background dark.

58. REQUESTIES
A darkground condenser
High intensity lamp
Funnel stop
Darkground condenser:
- Incorporates concentric reflecting mirror.
- A central one prevents light rays passing directly up
through the objective
-The other reflects the rays inwards on to the specimen at
a oblique angle.

59. FUNNEL STOP:
• Reduces the numerical aperture to less than
one.
• Small funnel shaped piece of metal or plastic .
• Special oil immersion fluorite objective can be
used with out funnel stop.

60. THE SLIDE:
• Should be 1.0-1.1mm
• Should be clean and free from grease.
THE FILM:
• Thin, so that moving objects are kept in one plane and
the background is dark.
• Thick - contrast is diminished
- objects move in and out of focus.
• The concentration of bacteria in the fluid specimen
must not be too great for an excessive number of
particles will scatter the light.

62. Uses of DGM
1. Examination of lightly stained prepared slide.
2. Examination of live and unstained preparation.
3. Determination of motility in cultures.
4. To demonstrate T.pallidium.
5. To detect leptospira and Borrelia in clinical
material.
6. To detect thin cell organelles such as flagella.

63. Disadvantage:
-Slides and coverslips should not be too thick.
-Condenser should be properly focused.
-Lighting should be sufficiently intense.
Advantage:
• Simple
• Conventional.

65. PHASE CONTRAST MICROSCOPY (PCM)
• Phase contrast microscopy is a contrast-
enhancing optical technique that can be
utilized to produce high-contrast images of
transparent specimens such as living cells,
microorganisms, thin tissue slices
• It enables us to observe unstained living
organism with good contrast and resolution.

66. Principle:
• Light rays passing through a transparent
specimen emerge as either direct rays or
diffracted rays.
• Phase contrast microscopy translates differences
in light in phases within the specimen into
differences in light intensities that results in
contrasts.

67. • this effect is amplified by using a microscope
equipped with a special annulus (below the
stage) and phase plates (located in the
objectives) which accentuate the phase changes
produced by the specimen.
Requisites:
1. High intensity illumination through an annular
diaphragm
2. Special phase objectives
3. Auxillary telescope

68. • A major advantage of phase contrast
microscopy is that living cells can be examined
in their natural state without being killed,
fixed, and stained

69. he phase-plate increases the
ase difference to half a wavelength

70. Phase diff
1/4
Phase diff
1/4
Total phase
diff=1/2

71. Image with regular brightfield
objectives Same image with phase contrast
objectives

72. Uses of PCM
1. Phase contrast is preferable to bright field microscopy
when high magnifications (400x, 1000x) are needed and
the specimen is colorless or the details so fine that color
does not show up well.
2. Cilia and flagella, are nearly invisible in bright field but
show up in sharp contrast in phase contrast.
3. Amoebae look like vague outlines in bright field, but
show a great deal of detail in phase.
4. Most living microscopic organisms are much more
obvious in phase contrast.

73. ADVANTAGES:
• Increase contrast without destroying sample.
• Specimen can remain alive.
• Moderate cost.
DISADVANTAGES:
• Limited in magnification and resolution.
• Only single cell/thin layers of cell is observable.

75. FLUORESCENCE MICROSCOPE (FM)
• Microscope used to study properties of organic or
inorganic substances using the phenomena of
fluorescence and phosphorescence instead of, or
in addition to, reflection and absorption.
• When certain material eg: oil/fat/dye exposed to
UV radiation- converts this invisible short waves
into visible longer wave length- becomes
luminous and are said to flourescence

76. TYPES OF Fluorescence:
• PRIMARY(AUTOFLUORESCENCE): Substance
fluorescing naturally e.g. vit A, porphyrin
• SECONDARY : production of fluorescence by
the addition of dyes (fluorochromes).

77. • The excitation light is emitted from mercury vapour
lamp
• An excitation filter passes light of desired wave length
to excite the fluorochrome – used to stain specimen
• The mirror reflects UV radiation of required wave
length down the microscope tube, through objective
on to the specimen
• The visible light from the specimen then passes back
through the objective to the dichroic mirror which
transmits it through a secondary filter to the eye piece.
PRINCIPLE:

78. • Barrier filter in the objective lens prevents
the excitation wavelength from damaging
the eye of the observer
• Fluorescing object appear bright against dark
back ground

81. STAINING TECHNIQUES:
1. Fluorochroming:
 Fluorescent dye used
 Non specific
Eg: Acridine Orange, Auramine rhodamine,
Calcofluor white.
1. Immunofluorescence:
 Fluorescent dye linked to specific Abs
 Specific
Eg: Fluoresecene isothiocyanate

82. LIGHT SOURCE:
• Mercury vapour lamp- better
• Xenon gas lamp
• Halogen filament lamp
• Enclosed in a protective housing as there is a risk of
explosion.
FILTERS:
• Primary filter [Excitation filter]-close to the lamp.
• Secondary filter [Barrier filter]-placed in the eye piece.

83. CONDENSER:
• Specimen stained with a fluorochrome dye ,a
bright ground three lens aplantic condenser
is used.
• Specimen stained with a fluorescent
antibody a dark ground condenser is used.

84. ILLUMINATION
1. Transmitted UV illumination-UV radiation
is transmitted from below through a sub
stage condenser.
2. Incident UV illumination-UV radiation is
transmitted from above through the
objective.
– directed horizontally from the lamp onto a
dichoric mirror set at 45o
in the microscope
tube.

85. OBJECTIVES
• Achromats are preferred to apochromates as
they rarely fluorescence.
• A high numerical aperture is preferred to
ensure maximum transmission of fluorescent
light from the objective.

87. Uses of FM:
• Immunofluoroscence technique detects and
identifies both etilogical agent and host
antibodies
• Fluorescent acid fast stain used for efficient
diagnosis of tubercle bacilli and to detect
cryptosporidium parvum.
• Acridine orange vital stain used in
parasitology[malaria,filaria]

88. ADVANTAGES:
• Very sensitive and specific.
• Sample can remain alive.
• Conventional.
DISADVANTAGES:
• Photo bleaching.

90. LOW POWER MICROSCOPES
Light microscope with low magnification eg.x4 tox50
• TYPES:
1. Stereoscopic microscope: for examining the detailed
morphology of colonies on culture plate.
2. Inverted microscope:
– lamp and the condenser are above the stage
– The objective, body and eye-piece are below the stage.
– Examination of virological cell culture.

91. ELECTRON MICROSCOPE (EM)
• An electron microscope is a type of microscope that
uses electrons to illuminate a specimen and create an
enlarged image.
• Electron microscopes have much greater resolving
power than light microscopes and can obtain much
higher magnifications.
• The first electron microscope prototype was built in
1931 by the German engineers Ernst Ruska and Max
Knoll

93. Principle:
• The electrons themselves are generated by a thin wire in
the "gun" of the electron microscope.
• Electricity is then passed through the wire and then
focused by magnets onto the object.
• When the electrons strike the object wrapped in gold,
they bounce off of the gold layer to a detector. This
forms a detailed image of the object.

94. Types of EM
• TRANSMISSION ELECTRON MICROSCOPE
(TEM)
• SCANNING ELECTRON MICROSCOPE (SEM)

95. TEM
• a beam of electrons is transmitted through a
specimen, then an image is formed, magnified
and directed to appear either on a fluorescent
screen or layer of photographic film, or to be
detected by a sensor such as a CCD camera.

97. A section of a cell of Bacillus subtilis, taken with a TEM.

98. SEM
• Produces images by detecting low energy
secondary electrons emitted from the surface
of the specimen due to excitation by the
primary electron beam
• SEM uses moving electron beam to scan the
specimen surface generating pictures with
great depth and 3 dimensional.

99. Scanning electron micrograph of E.coli.

100. USES:
• Enables the detailed study of
viruses,cells,fungal spores and tissues.
Advantages:
• High resolution and magnification
• Localisation of specific molecules can be
visualised.

101. Disadvantages:
• Expensive
• Technically challenging.
• Must coat sample with heavy metal to
visualise.

102. THANK YOU