This document provides information about optical microscopy and the components and functioning of microscopes. It discusses the basic types of microscopes as simple and compound. For compound microscopes, it describes the key optical components including objective lenses, eyepieces, condensers, and illumination sources. It explains how these components work together to produce magnified and resolved images. The document also covers topics like aberrations, numerical aperture, microscope heads and beam splitting. The overall purpose is to explain the basic principles of microscopy and identify the important parts of the microscope.

1. INDIAN DENTALACADEMY
Leader in continuing Dental Education

2. ♣ MICROSCOPE Basics and Beyond by
Mortimer Abramowitz Fellow, New York
Microscopical Society For Olympus America Inc.
Vol. I
♣ OPTICAL MICROSCOPY
Michael W. Davidson
(National High Magnetic Field Laboratory,The
Florida State University)
and
Mortimer Abramowitz(OlympusAmerica, Inc.,
2 Corporate Center Dr., Melville, NewYork
11747,)
♣ Theory and Practice of Histological techniques – John D
Bancroft & Marilyn Gamble
References:

3. At the end of the seminar, learner should be able to
♣ Know about basic keyword of optics and Microscope
♣ Know about types of microscope
♣ Know about components of microscope
♣ Know about function of components of microscope
♣ Know about principle mechanism of Binocular microscope

4. Keywords :
♣ LENS:
A piece of glass or other transparent
substance with curved sides for
concentrating or dispersing light rays.
♣ Types of lenses:
♣ Biconvex lens
♣ Biconcave lens
♣ Plano convex lens
♣ Plano concave lens
♣ Convex-concave lens
♣ Meniscus
♣ Compound lens

6. ♣ Retardation and Refraction
♣ Media through which the light is able to pass
will slow down the speed of the light is called
as Retardation.
♣ It does not deviate the light , if light enter
perpendicular to the surface of media.
♣ If the light enters the media through an angle
it slow down the speed (retardation) as well as
cause deviation (Refraction) of the light.

8. Refractive index:
♣ Basically it shows the density of the medium
its value is ratio of the SINS of angle of
incidence and angle of refraction.
♣ Denoted by RI and RI = sin i / sin r
♣ Higher the RI , denser the medium and more
of the light that enters the medium deviates
towards the normal.

9. RI = Sin i / Sin r
i
r

10. Image formation
♣ Parallel rays entering a lens are bought to
common single point by refraction this point is
called as Principal focus or Focal Point
♣ It is the point where clear image is formed
♣ Distance between center of lens and focal point
is known as Focal length of the lens

11. ♣ Along with principal focus ,lens also have some
other points known as Conjugate foci.
♣ Placing object on these points will form the
image over the screen on other side of the lens.
♣ Conjugate point will vary in position.
♣ As the object moves near to lens the image will
form further away, magnified and also inverted.
♣ This image is called as Real image.
♣ In microscope it is formed by Objective lens

14. ♣ If the object is placed yet nearer the lens and
with in the Principal focus , the image is formed
on the same side of the object.
♣ The formed image is enlarged , and right up (not
inverted), and cannot projected on the screen.
♣ This is called as Virtual image and is formed by
the eye piece of the compound microscope.
♣ Eye piece lens of the microscope forms the
enlarged Virtual image of the Real image formed
by the objective.

16. Aberration in lenses :
Chromatic aberration:
(Achromatism or Chromatic distortion)
ø A type of distortion in which there is a
failure of a lens to focus all colours to the
same convergence point.
ø It occurs because lenses have a different
refractive index for different wavelengths
present in a white light (the dispersion of
the lens).
ø Usually seen in red and blue wavelength

17. ♣ Blue light is refracted to the greatest extent
followed by green and red light.

18. ♣ Despite longitudinal (or axial) chromatic
aberration correction, apochromat objectives also
exhibit another chromatic defect.(Lateral
Chromatic aberration.)
♣ Even when all three main colors are brought to
identical focal planes axially, the point images of
details(specimen), near the periphery of the field
of view, are not of the same size

19. ♣ e.g., the blue image of a detail is slightly larger
than the green image or the red image in white
light, thus causing color ringing of specimen
details at the outer.
♣ This aberration is called Lateral Chromatic
aberration.
♣ Can be corrected by the eyepiece with
compensating lenses having opposite chromatic
magnification.

20. ♣ Chromatic aberration manifests itself as
"fringes" of color along boundaries that
separate dark and bright parts of the image

21. Spherical aberration:
♣ Spherical aberration is an optical effect
observed in an optical device (lens, mirror, etc.)
that occurs due to the increased refraction of
light rays when they strike a lens corner or
edges in comparison with those that strike
nearer the centre.

22. ♣ In lens systems, the effect can be minimized
using special combinations of convex
and concave lenses , as well as using aspheric
lenses.
♣ Advanced glass formulations that contain
materials such as fluorspar or newer synthetic
substitutes
♣ By limiting the outer edges of the lens from
exposure to light ,using diaphragms.

23. ♣ Numerical aperture:
♣ It is the ability of an objective to include or
grasp the various rays of light coming from each
illuminated part of the specimen and is directly
related to the angular aperture of the lens.
♣ It is also related to the ability of the lens to
resolve details; as high magnification require high
resolution, which is obtained with high numerical
aperture.
♣ NA(numerical aperture) = n X sin µ

24. ♣ NA(numerical aperture) = n X sin µ
♣ n is the refractive index of the medium
present between the specimen and surface of
the lens
♣ µ is the half of the angular aperture of the
lens.
Or
Angle between the optical axis of the lens
and outer most ray that can enter the lens
face.

29. A microscope is an instrument designed to make fine
details visible.
The microscope must accomplish three tasks:
♣ Produce a magnified image of the specimen
(Magnification)
♣ Separate the details in the image
(Resolution),
And
♣ Render the details visible to the eye, camera, or other
Imaging device
(Contrast).

30. ♣ First microscope was discovered by
Antony van Leeuwenhoek
first person to observe & describe microorganisms
accurately.
♣ Antony van Leeuwenhoek & Robert Hook
made improvements by working on lenses

31. Robert Hook constructed (1665) microscope
consisting of an objective lens , field glass & eye glass.

32. Microscopes are basically of two types
♣ Simple microscope
♣ Compound microscope

33. ♣ Simple microscope :
 More than five hundred years ago, simple glass
magnifiers were developed.
 These were convex lenses ( thicker in the centre
than the periphery ).
convex lenses

34. ♣ Simple microscope :
 The specimen or object could be focused by use
of the magnifier placed between the object and
the eye.
 These “simple microscopes”, along with the eye
lens, could spread the image on the retina by
magnification through increasing the visual angle
on the retina.

35. ♣ Simple microscope :

36. ♣ Compound microscope :
 Around the beginning of the 1700`s, through work
attributed to the Janssen brothers in the
Netherlands and Galileo in Italy, the compound
microscope was developed.

37.  In its basic form, it consisted of two convex
lenses aligned in series:
 An object glass (objective) closer to the object or
specimen,
and
 An eyepiece(ocular) closer to the observers eye
with screws of adjusting the position of the
specimen and the microscope lenses.
♣ Compound microscope :

38. ♣ Compound microscope :
 The compound microscope achieves a two-stage
magnification.
 The objective projects a magnified image into
the body tube of the microscope and the
eyepiece further magnifies the image projected
by the objective
For example,
the total visual magnification using a 10X
objective and a 15X eyepiece is 150X.

39. ♣ Compound microscope :
light pathway ray diagram

40. ♣ Compound
microscope :

41. ♣ A Compound microscope basically has two
component
♣ Optical component
♣ Mechanical component

42. ♣ Optical component include:
♣ Objective lens
♣ Eye piece lens
♣ Condenser
♣ Body lens
♣ Illumination source
♣ Beam splitter prisms
♣ Field lens

43. Optical
Component :

44. ♣ Mechanical component include:
♣ Stand
♣ Base
♣ Stage
♣ Nose piece
♣ Coarse and fine focusing knobs
♣ Diaphragm (Iris of microscope)
♣ Condenser holder

46. ♣ Optical component include:
♣ Objective lens
ø Objectives are the most important components
of the microscope.
ø Modern objectives, made up of many glass
elements, have reached a high state of
quality.
ø Now a days it is available as
ø Achromatic
ø Fluorite (semi-apochromatic)
ø Apo-chromatic

47. ♣ Achromatic objective lens:
ø The least expensive and most common
objectives
ø Correct the axial chromatic aberration in two
wavelengths ( RED & BLUE ) that are brought
into the same focus.
ø Further, they have to corrected for spherical
aberration.

48. ø The limited correction of achromatic objectives
leads to problems with colour microscopy and
Photomicrography.
ø When focus is chosen in the red-blue region of
the Spectrum, images will have a green halo (often
termed Residual colour).
ø Achromatic objectives yield their best results with
light passed through a green filter.
ø The lack of correction for flatness of field (field
curvature) further hampers achromat objectives.

50. ♣ Fluorite objective lens:
ø The next higher level of correction and cost is
found in objectives called fluorites or semi-
apochromats.
ø Fluorite objectives are produced from advanced
glass formulations that contain materials such as
fluorspar or newer synthetic substitutes .
ø These new formulations allow for greatly improved
correction of optical aberration.

51. ø Similar to the achromats, the fluorite objectives are
also corrected chromatically for red and blue light.
In addition, the fluorites are also corrected
spherically for these two colors.
ø The superior correction of fluorite objectives
compared to achromats enables these objectives to
be made with a higher numerical aperture, resulting
in brighter images.
ø Fluorite objectives also have better resolving power
than achromats and provide a higher degree of
contrast, making them better suited than achromats
for color photomicrography in white light.

53. ♣ Apochromatic objective lens:
ø The highest level of corrections (and expense) is
found in apochromatic objectives.
ø These objectives are corrected chromatically for
four colors,
ø deep blue
ø blue
ø green
ø red
ø They are spherically corrected for two or three
colors: deep blue, blue and green.

54. ø Apochromats are the best objectives for color
recording and viewing. Because of their high level
of correction.
ø Because of their high level of correction, such
objectives have, for a given magnification, higher
numerical apertures than do achromats or
fluorites.

55. ♣ Optical component :
♣ Eye piece lens
ø Also known as Occulars
ø Eyepieces work in combination with microscope
objectives to further magnify the intermediate
images so that specimen details can be observed.

57. ♣ There are two major types of eyepieces that are
grouped according to lens and diaphragm
arrangement:
♣ Negative eyepieces : with an internal diaphragm
between the lenses,
♣ Positive eyepieces : that have a
diaphragm below the lenses of the
eyepiece.
.

59. ♣ Negative eyepieces have two lenses:
♣ Commonly known as Huygenian eye piece
♣ The upper lens, which is closest to the
observers eye, is called the eye-lens
♣ The lower lens (beneath the diaphragm) is
often termed the field lens.
♣ In their simplest form, both lenses are plano-
convex, with convex sides facing the specimen

60. ♣ Positive eyepiece
♣ In this diaphragm below its lenses, commonly
known as the Ramsden eyepiece.
♣ This eyepiece has an eye lens and field lens
that are also plano-convex
♣ But the field lens is mounted with the curved
surface facing towards the eye lens.

61. ♣ More advanced eyepiece designs resulted in the
Periplan eyepiece which contains seven lens
elements that are cemented into a doublet, a
triplet, and two individual lenses.
♣ Design improvements in periplan eyepieces lead
to better correction for
♣ Residual lateral chromatic aberration,
♣ Increased flatness of field,
♣ And a general overall better performance when
used with higher power objectives.

62. ♣ Optical component :
♣ Condensers:
♣ Substage position
♣ Gathers light from the microscope light source
♣ Concentrates it into a cone of light that
illuminates the specimen with parallel beams of
uniform intensity from all azimuths over the
entire viewfield.

65. ♣ Specifically, appropriate use of the adjustable
aperture iris diaphragm (incorporated into the
condenser or just below it) is most important in
securing correct illumination, contrast, and depth
of field.
♣ The opening and closing of this iris diaphragm
controls the angle of illuminating rays (and thus
the aperture) which pass through the condenser,
through the specimen and then into the objective.

67. ♣ Types of codensers:
♣ A simple two-lens Abbe condenser
♣ The next highest level of condenser
correction is split between the Aplanatic and
Achromatic.
♣ Aplanatic-Achromatic condenser

69. ♣ The simplest and least corrected condenser is
the Abbe condenser that, in its simplest form,
has two optical lens elements
♣ Disadvantage:
ø Produce an image which is not sharp
ø Image is surrounded by blue and red color
at the edges.
♣ Advantages
ø Wide cone of illumination
ø Ability to work with long working distance
objectives.

70. ♣ The next higher level of condenser correction is
split between the aplanatic and achromatic
condensers that are corrected exclusively for
either
ø Spherical (aplanatic)
or
ø Chromatic (achromatic) optical aberrations.
♣ Achromatic condensers typically contain four lens
elements and have a numerical aperture of 0.95.

71. ♣ The highest level of correction for optical
aberration is incorporated in the Aplanatic-
Achromatic condenser.
♣ Advantage: This condenser is well corrected for
both
ø Chromatic
and
ø Spherical aberrations.
♣ A typical aplanatic-achromatic condenser
features eight internal lens elements cemented
into two doublets and four single lenses.

72. ♣ A critical factor in choosing substage condensers
is
♣ The numerical aperture,
ø Which will be necessary to provide an
illumination cone adequate for the
objectives.
ø The condenser numerical aperture
capability should be equal to or slightly less
than that of the highest objective numerical
aperture.

75. ♣ Microscope heads come in three types,
♣ Monocular,
♣ Binocular
♣ Trinocular.
♣ These designs all address the same problem,
how to compensate for the distance between
different peoples eyes.

76. ♣ A binocular head works by taking one light beam
and splitting it into two parts, that then go to the
eyes.
♣ The prism that does this is called the splitter
prism.
♣ Once the light leaves the splitter prism the
problems begin.
♣ As this distance (between eyes) changes ,the
tube length of the microscope changes.
♣ If this is not corrected the microscope will not be
parfocal.

77. ♣ Binocular and trinocular heads come in different
types of head:
♣ Compensating
♣ Siedontoff.
♣ A compensating head has a mechanism in it to
move the eyepiece tubes as the distance between
the eyepieces is changed.
♣ The Siedontoff binocular head solves the problem
by swiveling the eyetubes around the splitter
prism.

78. ♣ This allow the distance to change without
changing the tube length.
♣ This type of head must have two focusing
eyepieces or tubes for best results.
♣ When equipped this way the microscope should be
perfectly parfocal.
compensating head
Siedontoff head

79. ♣ Splitting the beam :
♣ In the binocular microscopy Splitting of the
single beam of light into two , is the most
important phenomenon.
♣ This process split the single beam coming from
the objective in to two for two separate eye
piece.
♣ To split the beam coming from the objective
lens, various companies uses different Prism and
Beam Splitter.

81. ♣ Prisms and beam splitters are essential
components that
♣ bend,
♣ split,
♣ reflect,
♣ fold
the light through the pathways of both simple and
sophisticated optical systems.
♣ Prisms are polished blocks of glass or other
transparent materials which is cut and ground into
specific tolerances and exact angles.

82. ♣ Prism can be employed to
♣ Deflect a light beam,
♣ Deviate a light beam
♣ Rotate or invert an image,
♣ Separate polarization states,
or
♣ Disperse light into its component
wavelengths
♣ Many prism designs can perform more than one
function, which often includes
♣ changing the line of sight
♣ shortening the optical path, thus reducing the
size of optical instruments.

84. ♣ Beam splitter
♣ Are utilized to redirect a portion of a light
beam while allowing the remainder to continue
on a straight path.
♣ Beam splitters can be
♣ As simple as a square or rectangular sheet
of glass coated with reflective material
or
♣ They can be integrated as surface
coatings into complex multi-element
optical assemblies.

85. ♣ The most common beamsplitter design enlists
two right-angle prisms that are coated on the
hypotenuse to produce a semi-reflective
surface, and then cemented together to form a
cube.

86. ♣ Types of Beam splitter:
• Cube beam splitter
• Pellicle beam splitter
• Perforated beam splitter