A presentation on microscopes- its evolution, history, uses, types, etc. beneficial for pathology students. impart knowledge about types of lens, parts of microscope ands their use.

1. BY
SWATI ARORA

2. INTRODUCTION

3. “FOR WHAT A BETTER ,FITTER,GIFT
COULD BE IN THIS WORLD’S AGED
LUCIOSITY?
TO HELP OUR BLINDNESS SO AS TO
DEVIZE A PAIR OF NEW AND
ARTIFICIAL EYES”
BY HENRY POWER(1661)

4. The invention of the microscope is
variously accredited to zaccharias
Jansen , a dutch spectaclemaker in
1590 and their device was the first
compound microscope. Others
known for their discoveries are
Antonvan leeuwenhoek; who built
the simple microscope and is
known as the FATHER OF
MICROSCOPY.

5. •Robert Hook, Marcello
Malpighi, Ernst Ruska and many
more and application of all
these microscopes to
physiological , pathological ,
therapeutical and
medicojurdicial purposes has
been very succesful.

6. DEFINITION
 The word microscope is derived from a
Greek word (micron – small,scopos-
aim) which means to look at small
objects.

7.  ( According to clay and court, 1975 ) -
 “ If we consider a microscope to be an
instrument by which we can observe
objects or parts of objects which are too
minute to be visible to the naked
eyes,and which can be used to
investigate minute structures of plants
or animals and thus bring to our
knowledge facts not otherwise
ascertainable, then the microscope is a
comparatively modern invention and
dates back only to about the end of the
sixteenth century”

8. HISTORY OF MICROSCOPE
 CIRCA 1000 AD – The first vision aid was invented
called a reading stone.
 CIRCA 1284- Italian salvinoD’Armate is credited
with inventing the wearable eye glasses.
 1590- Two dutch spectacle maker, Zaccharias
Janssen and his father Hans starting
experimenting with lenses. They put several
lenses in a tube and made a very important
discovery. They had invented the compound
microscope.

9.  1665 – English physicist, ROBERT HOOK
looked at a sliver of cork through a
microscope lens and noticed some “pores”
or “cells” in it.
 Also known as the ENGLISH FATHER OF
MICROSCOPY.

10.  1674 – ANTONIEVAN LEEUWENHOEK built
a simple microscope with only one lens to
examine blood , yeast and many other tiny
objects.
 He was the first person to describe tiny
cells and bacteria.
 18 CENTURY- Technical innovations
improved microscope lenses combining
two types of glass reduced the “chromatic
effect”.

11.  1872 – ERNST ABBE, the research director
of the zeiss optical works provided
calculations that allowed for the maximum
resolution in microscope possible.
 1903 – RICHARD ZSIGMONDY developed the
ultramicroscope that could study objects
below the wavelength of light.

12.  1932 – FRITS ZERNIKE invented the phase
contrast microscope that allowed for the study of
colourless and transparent biological materials.
 1931 – ERNST RUSKA co-invented the electron
microscope
 1981 – GERD BINNIG and HEINRICH ROHRER
invented the scanning tunneling microscope
 It is the strongest microscope to date.

13. THE LENS
 The word lens is derived from the latin word
“lentil”because they resemble the shape of
a lentil bean.
 EVOLUTION OF LENS-Egyptians knew and
practiced the art of cutting and polishing of
stones. From egyptians this art was
extended to the greece and itlay.Egyptians
artifacts include rock crystals in the form
of convex lenses.The greeks and romans
continued with these type of lenses up to
the end of the roman empire.

14.  Knew and practiced the art of glass
blowing.
 Observed that objects placed in a bulb
filled with water appeared magnified.

15. DEFINITION
 A lens is a 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.

16. TYPES OF LENSES
 Two types of lenses –
 A) POSITIVE LENS- concentrate light rays
or converge to form real image.
 Are thicker at the centre than at periphery.

17. B) NEGATIVE LENS- Diverge or scatter
light rays.
Do not form real image.
Thinner at the centre.

18. PROPERTIES
A) RETARDATION – Media through which
light is able to pass will slow down the
speed of the light in proportion to the
density of the medium. This is called as
retardation.
 The higher the density more will be the
retardation.

19. REFRACTION
 When light rays enter the glass at an angle,
a deviation of direction will occur in
addition to the retardation called as
refraction.
 A curved lens will exhibit both retardation
and refraction.

20.  The degree of refraction is governed by-
 The angle of incidence.
 The density of the glass.(its refractive
index)
 The curvature of the lens.

21. REFRACTIVE INDEX
 The ratio of the sine values of angle of
incidence(i) and refraction(r) gives a figure
known as refractive index(RI).
 RI=sine i/sine r
 It is the ratio of velocity of light in air to
the velocity of light in that substance.

22.  The greater the refractive index, the
higher the density of the medium.
 Air has RI= 1.00 ,
 Water= 1.30
 Glass= average of 1.5

23. ANGLE OF INCIDENCE – The angle at
which light strikes the lens.
ANGLE OF REFRACTION- The angle
to which rays are deviated within the
glass or other transparent medium.
CRITICAL ANGLE- Is the angle of
incidence above which the total
internal reflection occurs.

24. TOTAL INTERNAL REFLECTION
 When the angle of incidence is greater (i.e.
the ray is closer to being parallel to the
boundary) than the critical angle – than the
light will stop crossing the boundary
altogether and should be totally reflected
back internally.
 This occurs only when light travels from a
medium with a higher refractive index to
one with a lower refractive index.

25. FOCAL POINT
FOCAL PLANE
FOCAL LENGTH / FOCAL
DISTANCE

26. CONJUGATE FOCI
REAL IMAGE
VIRTUAL IMAGE

27. •PARFOCAL DISTANCE

28. FOCAL DEPTH- Depth of the specimen
layer which is in sharp focus at the
same time, even if the distance
between the objective lens and the
specimen plane is changed when
observing and shooting the specimen
plane by microscope.

29. MAGNIFICATION
 Is the number of times an image size is
enlarged where size is measured in the
degree of an angle formed by lines running
from either end of the image to the vertex
at the observer’s eye.

30. Total magnification is the product of the
magnification values of the objective and
eyepiece with standard optical tube length of
160mm.
Thus, the formula is –
Optical tube length
focal length of objective
TOTAL MAGNIFICATION

31. EMPTY MAGNIFICATION
 Exceeding the limit of useful magnification causes
the image to suffer from the phenomenon of empty
magnification.
 Increasing magnification through the eyepiece or
intermediate tube lens only causes the image to
become more magnified with no corresponding
increase in detail resolution.

32. RESOLUTION
 It is the smallest distance between
two dots or lines that can be seen as
separate entities.
 Restricted by the two factors-
 Numerical aperture of the lens
 Wavelength of the light employed.
 So, Resolution= 0.61

33. RESOLVING POWER
 The resolving power of the lens is its ability
to resolve the detail that can be measured
 As numerical aperture increases , resolving
power increases.
 Working distance , flatness of field and
focal length decreases.

34. ILLUMINATION
The application of light onto an object
or specimen under a microscope.
Artificial illumination supplied by
electric lamp is most commonly used.
The lamp may be a simple pearl bulb
or high intensity lamp used in
conjugation with a condensor and an
iris diaphragm.

35.  The source of illumination should be –
 Uniformly intense
 Should completely flood back lens of the
condensor with light when the lamp iris
diaphragm is open.
 Make the object appear as though it were
self luminous

36. METHODS FOR ILLUMINATION
a) CRITICAL ILLUMINATION BY
NELSON.
b) KOHLER ILLUMINATION.

37. CRITICAL ILLUMINATION

38. KOHLER ILLUMINATION

39. MICROMETRY
 Standard unit of measurement in
microscopy is micron , which is 0.001mm.
 To measure microscopic objects an
eyepiece micrometer scale is used in
conjugation with stage micrometer.

40. FAULTS OF LENS
 Three major classes of lens errors are -
 ON- AXIS ERRORS
 OFF- AXIS ERRORS
 GEOMETRICAL DISTORTION

41.  A)ON- AXIS ERRORS ARE –
 CHROMATIC ABERRATION
 SPHERICAL ABERRATION
 B) OFF- AXIS ERRORS ARE-
 COMA
 ASTIGMATISM
 FIELD OF CURVATURE
 C) GEOMETRICAL DISTORTION IS OF TWO TYPES –
 PIN CUSHION (POSITIVE) DISTORTION
 BARREL(NEGATIVE) DISTORTION

42. CHROMATIC ABERRATION
 Most common fault observed in spherical
lenses.
 Occurs because the lens refracts the
various colour present in white light at a
different angle according to wavelength.

43.  This type of aberration can be reduced or
eliminated by making compound lenses
composed of individual elements having
different colour dispersing properties.
 The correction of this fault is called as
ACHROMATISM.
 ACHROMAT- Corrected for two colours , blue
and red
 APOCHROMAT- Corrected for three or more
colours(i.e.for secondary spectrum of yellow/
green)
 Has less spherical aberration.

44. SPHERICAL ABERRATION
 It is due to the use of lenses having
spherical curvature.
 Occurs when light waves passing through
the periphery of lens are not brought into
exact focus with those passing through
the centre.
 It is corrected by making combination of
lens elements of different glass.
Example- Flourite and of differing
shapes.

45. OFF – AXIS ERRORS
 a) COMA- Most commonly encountered with off
axis light rays when microscope is out of proper
alignment.
 It is named for its strong resemblance to the shape of
a comet tail.
 Manifested by a streak of light that appears to
emanate from a focused spot at the periphery of
viewfield.
 The distinct shape displayed by images suffering from
coma aberration is the result of refraction differences
by light rays through various lens zones as the incident
angle becomes more oblique.(off-axis)

47. ASTIGMATISM
 When image of an off axis object point
is not focused to a single point but
separated to a concentric line image
and radial line image is called as
“astigmatism.”
 Depends more strongly on the oblique
angle of the light beam.
 Can be corrected or reduced by careful
alignment and adjustment of the
individual lens elements with spacers
and shims.

49. FIELD CURVATURE
 This aberration is the result of lenses that have
curved surfaces.
 When light is focused through a curved lens, the
image plane produced by that lens will be
curved.
 Produces an image plane having the shape of a
concave spherical surface(resembling a convex
lens surface); as seen from objective.
 Can be corrected by adding corrective lens
elements to the objective ( fiat field objective)
 These objective termed as plan or plano are the
most common type of objective in use.

51. GEOMETRICAL DISTORTION
 The two most prevalent type of distortion are-
 Manifested by changes in the shape of an
image rather than sharpness or colour
spectrum.
 POSITIVE( Pincushion) DISTORTION
 NEGATIVE(Barrel) DISTORTION
 Most severe in specimens that have straight
lines,such as periodic grids, squares,
rectangles, or other regular polygonal
features.

52.  Complex lens system have pronounced
distortion which may vary with focal length.
 Distortion is often found in compound
lenses containing meniscus ,concave,
hemispherical and thick convex lenses.
 Pincushion distortion- at long focal length
 Barrel distortion- at short focal length.

53. PARTS OF A MICROSCOPE
 The standard monocular microscope is
composed of two main parts-
 The MICROSCOPE PROPER- includes
body tube with the objective at one end
and eyepiece at the other.
 The STAND- includes supporting,
adjusting,and illuminating apparatus.

55. LIGHT SOURCE- An essential part of
the system.
At one time sunlight was the usual
source.
A progression was developed, from
oil lamps to the low voltage electric
lamp.
These operate via transformer and
can be adjusted to the intensity
required.
Some of the instruments have their
light source built into them.

56. THE MICROSCOPE PROPER
THE EYEPIECE( OR OCULAR)-
Final stage in the optical path of
microscope.
FUNCTION- is to magnify image
formed by the objective within the
body tube and present the eye
with the virtual image apparently
in the plane of the object being
observed

57.  The two most common eye pieces are-
 HUYGENIAN PATTERN
 COMPENSATING EYEPIECE
 Composed of two lenses-
 The LOWER OR FIELD LENS- collects
the image that have been formed by
the objective and cones it down to a
slightly smaller image at the level of
the field stop within the eye piece
 The UPPER LENS – produces an
enlarged virtual image.

58. THE OBJECTIVE
 Screws into the lower end of the body tube
by means of a standard thread.
 They are designated not by their magnifying
power but by their focal length (from 2mm
to 50mm).
 Within the objective there may be lenses
and elements five to fifteen in number
depending upon image ratio, type and
quality.

59.  FUNCTION- To collect maximum
amount of light possible from the
object, unite it and form a high quality
magnified real image.
 NUMERICAL APERTURE- The ability of
an objective to resolve detail is
indicated by its numerical aperture.
 Expressed as a figure and is engraved
on the body of the objective.

60. Calculated from the formula-
Numerical aperture = n x sin u,
where n= refractive index of the
medium between the coverglass
over the object and the front lens
of the objective . Example- Air,
Water and immersion oil.
u=is the angle included between
the optical axis of the lens and
the outermost ray which can
enter the front lens.

61.  Objectives are available in varying quality
and types-
 ACHROMATIC
 APOCHROMATIC
 PLANAPOCHROMATS
 PLANACHROMATS
 NOW A DAYS, Objectives are PARFOCAL
and PAR CENTRAL.

62.  Generally, there are four objectives in a
microscope, each with a different
magnifying power.
 a) 4x or SCANNING OBJECTIVE
 b) 10x or LOW POWER OBJECTIVE
 c) 40x or HIGH DRY OBJECTIVE

63.  d) 100x or OIL IMMERSION OBJECTIVE-
 Highest magnification on microscope.
 A drop of cedar wood oil is used on the
slide.
 Oil has refractive index identical to
that of glass. Thus, prevents refraction
of light rays,allowing the maximum
amount of light to be gathered by the
objective from the specimen.
 This increases the resolution of the
image.

64. EFFECTS OF A HIGH NUMERICAL
APERTURE
 Resolution of an objective is increased
 DISADVANTAGES-
 Depth of focus is reduced.
 Flatness of field is reduced. So, that
edges are out of focus.

65. NOSEPIECE OR CARRIER
 Fitted at the lower end of the body tube.
 Rotates on a central pillar and holds the
objective.
 Designated by the number of objective it
carries.
 For example- Double, Triple or Quadruple
nosepiece.

66. BODY TUBE
 Three main forms of body tube are-
 MONOCULAR
 BINOCULAR
 COMBINED PHOTO BINOCULAR
 The tube length should always be set to
the standard of 160mm, if a draw tube is
fitted.

67. SUPPORT,ADJUSTMENT AND
ILLUMINATION
 SUPPORTING STRUCTURE- Body tube is
attached to a limb, which is usually hinged to
a pillar or base
 Two types of adjustment-
 1) COARSE ADJUSTMENT- working by rack
and pinion, enables the body tube to be
moved up and down.
 2) FINE ADJUSTMENT- working by
micrometer screws and levers or cams.

68. OBJECT STAGE
 At lower end of the limb supporting the body
tube and adjustment is a platform or stage, on
which objects to be examined are placed.
 The stage should be sturdy and perpendicular
to the optical path.
 The stage is provided with a mechanical stage
that allows controlled movement in two
diections
 Circular rotating stages are also available.

69. ILLUMINATING APPARATUS
 Below the stage , there is an
adjustable substage
 Substage consist of-
 The condensor
 An iris Diaphragm
 A Filter carrier
 A mirror

70. THE CONDENSOR
First major optical component.
PURPOSE- To focus or concentrate
the available light into the plane of
object.
Within the comfortable limits, the
more light at the specimen, the better
is the resolution of the image.
In most of the cases,condensors are
provided with a adjustment screws for
centring the light path.

71.  Two – lens Abbe condensor is in common
use but is not very efficient.
 It should not be used with apochromatic or
fluorite objectives.
 To obtain perfect results with such
objectives, a three lens aplanatic or more
highly corrected chromatic condensor
should be used so as to get a crisp image
with good resolution.

72. THE IRIS DIAPHRAGM
 All condensors have an aperture diaphragm with
which the diameter of the light beam can be
controlled.
 Adjustment of the iris diaphragm will alter the size
and volume of the cone of light focused on the
object.
 Correct setting for the diaphragm is when the
numerical aperture of the condensor is matched
with the numerical aperture of the objective.
 If the diaphragm is closed too much- the image
becomes too contrasty and refractile.
 If diaphragm is left wide open- image will suffer
from glare due to extraneous light interference.

73. THE FILTER AND FILTER CARRIER
 The intensity of illumination should always
be reduced by using light absorbing
filters,or the rheostat of the lamp
transformer.
 Many condensors are fitted with swing out
top lens.This is turned into the light path
when the higher power objectivew are in
use.It focuses the light into a field more
suited to the smaller diameter of the
objective front lens.
 Filter carrier is a metal ring that facilitate
the easy removal of the filters.

74. THE MIRROR
Two sided mirror – plane on one side
and concave on the other is used.
Fitted about 4 inches below the stage.
Concave mirror has a focus ,since it
causes the light rays which has been
reflected, to converge together and
form an image.
It takes the place of a condensor
when used with low power
objective.since it requires a large
area of the object to be illuminated.

75. Plane mirror must always be used
with the condensor.
Plane mirror is used when light source
is distant one(natural daylight) –
parallel rays of light are reflected
parallel into condensor.
Concave mirror is usedwhen light
source is near the microscope
(example- electric lamp)- Divergent
light rays are converted into parallel
rays which are then directed into the
condensor.

76. TYPES OF MICROSCOPES
BASED ON THE LIGHT SOURCE –
OPTICAL MICROSCOPE
NON OPTICAL MICROSCOPE

77. OPTICAL MICROSCOPE-:
1) SIMPLE MICROSCOPE
2) COMPOUND MICROSCOPE

78. Other types of optical or light
microscope-
Dark ground microscope
Phase contrast microscope
Polarized microscope
Interference microscope
Confocal microscope

79. NON –OPTICAL MICROSCOPE
ARE-
 ELECTRON MICROSCOPE- :
1) SCANNING ELECTRON MICROSCOPE
2) TRANSMISSION ELECTRON
MICROSCOPE

80.  Other non- optical microscope are-
 SCANNING TUNNELING MICROSCOPE
 ACOUSTIC FORCE MICROSCOPE
 SCANNING HELIUM MICROSCOPE
 MAGNETIC FIELD MICROSCOPE
 SCANNING MAGNETIC FLUX
MICROSCOPE