general microscopy

1. MICROSCOPY
Mr. R. K. Lodha

2. What IsA Microscope ?
•A microscope (from the Ancient Greek: micro- "small“ and scope-"to
look") is an instrument used to see objects that are too small for the naked
eye.
•A microscope is an instrument that produces an accurately enlarged image
of small objects.
•The science of investigating small objects using such an instrument is called
microscopy.
•Microscopic means invisible to the eye unless aided by a microscope.

3. A microscope is an optical instrument that uses a lens or a combination of lens to
produce a magnified image of an object; too small to be seen with the naked eye.

9. Two Dutch eye glass makers, Zaccharias Janssen and son
Hans Janssen experimented with multiple lenses placed
in a tube.
The Janssens observed that viewed objects in front of
the tube appeared greatly enlarged, creating both the
forerunner of the compound microscope and the
telescope.
History Sequence…… 1590

10. • 1665 - Robert Hooke's book called
Micrographia officially documented a
wide range of observations through the
microscope.
• 1674 - Anton van Leeuwenhoek grinded
lenses to achieve greater magnification
which he utilised to make a microscope,
enabling detailed observations to be
made of bacteria.
Anthony van Leeuwenhoek
1632-1723
Robert Hooke
1635-1703
Hooke Microscope
• Anthony van Leeuwenhoek and Robert Hooke made
improvements by working on the lenses

11. History Sequence…… 1665

12. Anton van Leeuwenhoek built a simple
microscope with only one lens to examine
blood, yeast, insects and many other tiny
objects.
Leeuwenhoek was the first person to describe
bacteria, and he invented new methods for
grinding and polishing microscope lenses that
allowed for curvatures providing
magnifications of up to 270 diameters, the
best available lenses at that time.
History Sequence…… 1674

14. Technical innovations improved microscopes,
leading to microscopy becoming popular among
scientists.
Lenses combining two types of glass reduced
the "chromatic effect" the disturbing halos
resulting from differences in refraction of light.
History Sequence…… 18th century

15. • Joseph Jackson Lister reduces spherical aberration or the
"chromatic effect" by showing that several weak lenses used together at
certain distances gave good magnification without blurring the image.
• He created an achromatic lens to eradicating the chromatic effect
caused by different wavelengths of light.
• This was the prototype for the compound microscope.
History Sequence…… 1830

16. Ernst Abbe, then research
director of the Zeiss Optical
Works, wrote a mathematical
formula called the "Abbe Sine
Condition".
His formula provided calculations
that allowed for the maximum
resolution in microscopes
possible.
History Sequence…… 1872

17. He won the Nobel Prize in Chemistry in 1925.
History Sequence…… 1903

18. Ernst Ruska co-invented the electron microscope for which he won the
Nobel Prize in Physics in 1986.
• An electron microscope depends on electrons rather than light to
view an object, electrons are speeded up in a vacuum until their
wavelength is extremely short, only one hundred-thousandth that of
white light.
• It possible to view objects as small as the diameter of an atom.
History Sequence…… 1931

19. Ernst Ruska…… 1931

20. Frits Zernike invented the
phase-contrast microscope that
allowed for the study of
colorless and transparent
biological materials for which he
won the Nobel Prize in Physics
in 1953.
History Sequence…… 1932

21. Gerd Binnig and Heinrich Rohrer invented
the scanning tunneling microscope that
gives three- dimensional images of objects
down to the atomic level.
Binnig and Rohrer won the Nobel Prize in
Physics in 1986.
The powerful scanning tunneling microscope
is the strongest microscope to date.
History Sequence…… 1981

23. The first commersial microscopes
• 1939 Elmiskop by Siemens Company
Elmiskop I
• 1941 microscope by Radio corporation of America (RCA)
– First instrument with stigmators to correct for
astigmatism. Resolution limit below 10 Å.

25. Important Terms related to
Microscopy

27. The media through which the light passes will
be able to slow down or retard the speed of the
light in proportion to the density of the medium.
Higher the density, greater the retardation.
Light rays entering a sheet of glass at right angle are
retarded but their direction is unchanged.

28.  If the light enters the glass at any angle other than right angle, a
deviation in the direction will occur in addition to retardation,
known as Refraction.
 A curved lens will exhibit both refraction and retardation.
 The extent of which is determined by angle of incidence, refractive
index and curvature of the lens.

29.  Refraction is the bending of light as it passes from one medium to
another of different density.
 Immersion oil, which has the same index of refraction as glass, is
used to replace air and to prevent refraction at a glass-air interface.
 An example would be when one looks at objects just below the
surface of water in a pond or other body of water…..the objects
become refracted or “distorted” from the true image.

31. Diffraction is the change in the direction and the intensities of
group of waves after passing by an obstacle or through an aperture,
whose size is approximately the same as the wavelength of the wave.
Dispersion is a phenomenon, in which separation of light into its
constituent wavelength occurs from entering a transparent medium. For
example, white light consist consists of more than one wavelength. They
would be separated or they would distribute when they pass through
prism or certain other medium.
Interference is the variation of wave amplitude that occurs when
waves of the same or different frequency come together. There could be
constructive interference or there could be destructive interference.

32. RESOLUTION OF LENS
where  is the wavelength of the illumination,
n is the refractive index of the medium in front of the lens,
 is the semi-angle (aperture angle) subtended by the object at the lens
• Resolution defines the smallest separation of two points in the object, which
may be distinctly reproduced in the image.
The resolving power for light microscope is determined by
diffraction aberration and can be defined as
 
k
nsin
k is a constant usually taken to be 0.61.

33. Optical Microscope –
 = 50 nm (for white light Illumination)
n sin  = 0.135 (for an oil immersion lens)
Therefore, it is possible to achieve a resolution of
about 250 nm in Optical Microscopes.
• Filters can also be used to enhance the resolving
power of an objective. For light:
– The shorter wavelengths are at the violet-blue-green
end of the spectrum
– The higher wavelengths are at the orange-red of the
spectrum.

34. LIMIT OF RESOLUTION
• It is the smallest distance by which two objects can be separated and still be
distinguishable as two separate objects.
• It is given by formula
where, d = limit of resolution and
λ = wavelength of light
Which is, 0.40 μm per blue light (min in visible range)
0.70 μm per red light (max in visible range)
0.55 μm per green light

35. • Using the values 1.3 for NA and 0.55 μm, the wavelength of green light, for λ,
resolution can be calculated as follows-
d= 0.55/2X1.30
= 0.21 μm
From these calculations, we can conclude that the smallest details that can be
seen by the typical light microscope are those having dimensions of
approximately 0.2 μm.
• The greatest resolution in light microscopy is obtained with the
shortest wavelength of the visible light and an objective with the
maximum NA.

36. MAGNIFICATION
1000mm
35 mm slide
24x35 mm
35 mm
1000 mm
M = = 28
p The projected image is 28
times larger than we would see
it at 250 mm from our eyes.
If we used a 10x magnifier we would have a
magnification of 280x, but we would reduce the
field of view by a factor of 10x.
Rule of thumb
P is not to exceed 1,000
times the NA of the
objective

37. • Simple microscopes have only a single lens: Magnifying glass has a
limited magnification of 10x-20x.
• Modern microscopes magnify using both in the objective and the ocular
lenses and thus are called “compound microscopes”
• Total magnification of compound microscope
MTOT=MOBJX MINTX MEYEPIECE
• Objective magnification defined by focal lengths of tube lens and
objectives
• Mobj=ftl/fobj
• Tube lens has a standardized value for specific manufacture
• Zeiss, leica, olympus 165 mm, nikon 200 mm typical magnification.

38. USEFUL MAGNIFICATION RANGE
Mobj Meyepiece NAobj Mtot Museful Magnification
10x 10x 0.35 100 175-350 low
40x 10x 0.70 400 350-700 ok
100x 10x 1.40 1000 700-1400 ok
100x 15x 1.40 1500 700-1400 empty
• Microscope resolution is limited by NA and wavelength.
• Enlargement of image does not necessarily resolve new features.
• Excessively large magnification is called empty magnification.
• Useful magnification = 500-1000 x NA of objective

39. Magnification vs. Resolution
Magnification is how
much bigger a sample
appears to be under the
microscope than it is in
real life.
Resolution is the
ability to distinguish
between two points on
an image - the amount
of detail.
Increasing the magnification does not increase
the resolution of the image!!
Total Magnification = objective
magnification x eyepiece magnification
e.g. if two objects
are less than
200nm apart they
are seen as one
object.

40. NumericalAperture
• NA: Ability to gather light and resolve fine specimen detail
• Resolving power is directly related to numerical aperture.
• The higher the NA the greater the resolution
• Resolving power:
The ability of an objective to resolve two distinct lines very close
together
NA= n sin u
• N is the lowest refractive index between the object and first objective element
(hopefully 1)
• u is half the angular aperture of the objective

42. The wider the angle the lens is capable of receiving light at, the greater its resolving power
The higher the NA, the
shorter the working
distance
NumericalAperture

43. Numerical Aperture (NA)
•The angle α is the half-aperture angle, which is expressed as a
sine value.
•The sine value of the half-aperture angle multiplied by the refractive
index n of the medium filling the space between the front lens and
the cover slip gives the numerical aperture (NA).
• NA= n sine α
•With dry objectives the value of n is 1, since 1 is the refractive
index of air.
•When immersion oil is used as the medium, then n is 1.56 and if α
is 58°, the NA= n sine α = 1.33.

44. • The visible light range is between 400 nm (blue light) and 700 nm
(red light). Thus it is apparent that the resolving power of the
optical microscope is restricted by the limiting values of the NA
and the wavelength of the visible light.

45. WORKING DISTANCE
• Working distance may be defined as the distance between the front lens facing
object of the objective and the object on the slide.
• While increasing magnification working distance decreases.
• Commonly it is for,
10x = 9mm
45x = 0.3mm
100x = 0.1mm.

46. An optical aberration is a distortion in the image formed by an optical system compared to the
original. They can arise from the limitations of optical components such as lenses and mirrors.
• Spherical aberration-occurs in a spherical lens or mirror because these do not focus parallel
rays to a point, but instead along a line. Therefore, off-axis rays are brought to a focus closer
to the lens or mirror than are on-axis rays.
• Coma-occurs because off-axis rays no not quite converge at the focal plane. Coma is
positive when off-axis rays focus furthest from the axis, and negative when they are closest.
• Astigmatism- occurs in lenses because a lens has different focal lengths for rays of different
orientations, resulting in a distortion of the image. In particular, rays of light from horizontal
and vertical lines in a plane on the object are not focused to the same plane on the edges of
the image.
• Chromatic aberration- occurs in lenses because lenses bring different colors of light to a
focus at different points.
OpticalAberrations

47.  Spherical aberration is caused by the virtue of its curvature, where
the light rays entering the lens at the periphery are refracted more
than the light rays entering at the center of the lens, and thus not
brought to a common focus.
 These defects are also corrected by using a combination of lens
elements of different glass and of different shape.

48. SPHERICAL
ABERRATION
Generated by nonspherical wavefronts produced by the objective, and increased tube length,
or inserted objects such as coverslips, immersion oil, etc.
Essentially, it is desirable only to use the center part of a lens to avoid this problem.
F1 F2
F1
Corrected lens
It occurs in a spherical lens or mirror because these
do not focus parallel rays to a point, but instead
along a line. Therefore, off-axis rays are brought to
a focus closer to the lens or mirror than are on-axis
rays.

49. COMA
off-axis rays no not quite converge at the
focal plane. Coma is positive when off-
axis rays focus furthest from the axis,
and negative when they are closest.

50. Astigmatism
Perfectly symmetrical
image field is moved off
axis, it becomes either
radially or tangentially
elongated.

51. CHROMATIC ABERRATIONS
ChromaticAberration, also known as
“color fringing” or “purple fringing”, is
a common optical problem that occurs
when a lens is either unable to bring all
wavelengths of color to the same focal
plane, and/or when wavelengths of
color are focused at different positions
in the focal plane

52.  Light is composed of spectrum of colors, each having a different
wavelength, will be refracted to a different extent, with blue being
brought to a shorter focus than red.
 This lens defect is known as chromatic aberration and results in an
unsharp image and distorted edges & it’s correction is known as
achromatism.

53.  It is possible to construct compound lenses of different glass
elements to correct this.
 An achromat is corrected for two colors, red and blue,
producing a secondary spectrum of yellow/green, which is
in turn corrected by adding more lens components like
fluorospar, three colors can be brought into focus – the more
expensive – Apochromat.

55. Question
ocular power = 10x
low power objective = 20x
high power objective = 50x
a) What is the highest magnification you could get
using this microscope ?
Ans:500x
Ocular x high power = 10 x 50 = 500. (We can
only use 2 lenses at a time, not all three.)

56. b) If the diameter of the low power field is 2 mm,
what is the diameter of the high power field of
view in mm ?
Ans: 0.8 mm
The ratio of low to high power is 20/50.
So, at high power you will see 2/5 of the low
power field of view (2 mm).
Then, 2/5 x 2 = 4/5 = 0.8 mm
** if in micrometers ? 800 micrometers
Question

57. C) If 10 cells can fit end to end in the low
power field of view, how many of those cells
would you see under high power ?
Ans: 4 cells.
At high power we would see 2/5 of the low
field.
So, 2/5 x 10 cells = 4 cells would be seen under
high power.
Question

58. Anatomy of a Compound
Microscope

60. Parts of a Compound
Microscope

61. PARTS OFACOMPOUND MICROSCOPE
Microscope proper: Incorporating the body tube with the objective at one
end and the eyepiece at the other end.
The stand which include the supporting, adjusting and illuminating
apparatus

62. COMPONENT PARTS OFAMICROSCOPE
A. SUPPORT SYSTEM
(i) Base
(ii) Pillars
(iii) Handle/Limb
B. FOCUSSING SYSTEM
(i) Course adjustment screw
(ii) Fine adjustment screw
C. THE STAGE
(i) Fixed Stage
(ii) Mechanical stage

63. D. OPTICALSYSTEM
(i) Body Tube
(ii) Nose piece a.Fixed
b. Revolving
(iii) Objective lenses
(iv) Eye piece
E. ILLUMINATION SYSTEM
(i) Source of light
(ii) The mirror
(iii)The condenser

65. Lenses:
Ocular Lens: eyepiece lens
Objective Lens: can be low, medium or high power
Look at magnification on lens
Lower power is smaller in size

66. Letting in Light:
• Mirror or Illuminator:
directs light up through the
specimen
• Diaphragm: regulates
amount of light
– Disk with different sized
“iris” or openings

67. • Arm: connects stage and body
tube
• Stage: platform with opening
over which a specimen is placed
(clips to hold slide)
• Base: supports microscope

68. • Eyepiece (ocular): part you look
through, holds ocular lens,
magnifies 10x
• Body tube: connects eyepiece &
objective lenses
• Nosepiece: holds objective lenses
(can be turned)

69. Focusing:
Coarse Adjustment Knob:
use on low power only!!
(never use with high power
you can break your slide!)
Fine Adjustment Knob:
once low power is focused
switch to high power and use
fine adjustment.

70. CLASSIFICATION OF LIGHT MICROSCOPES
Depending on lens system:
Simple
Compound
Depending on optical technique:
Bright Field
Dark Field
Phase Contrast
Interference fluorescence

71. SIMPLE MICROSCOPE
Because of the limited ability of the eye's lens
to change its shape, objects brought very close to
the eye cannot have their images brought to focus
on the retina.
The accepted minimal conventional viewing
distance is 10 inches or 250 millimeters (25
centimeters).
SIMPLE MAGNIFIER. ASIMPLE
MAGNIFIER USES ASINGLE
LENS SYSTEM TO ENLARGE
THE OBJECT IN ONE STEP

72. More than five hundred years ago, simple glass magnifiers were
developed. These were convex lenses (thicker in the center than the
periphery).
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 cornea and eye lens, could
spread the image on the retina by magnification through increasing the
visual angle on the retina.

73. The "simple microscope" or magnifying glass
reached its highest state of perfection, in the 1600's, in
the work of Anton von Leeuwenhoek who was able to
see single-celled animals ("animalcules") and even some
larger bacteria.
The image produced by such a magnifier, held close
to the observer's eye, appears as if it were on the same
side of the lens as the object itself.
VON LEEUWENHOEK
MICROSCOPE. (CIRCA
LATE 16002)

74. Such an image, seen as if it were ten inches from the eye, is
known as a virtual image and cannot be captured on film.
These magnifiers had severe limitations in specimen positioning,
illumination, lens aberrations, and construction

75. LIGHT MICROSCOPY
THE COMPOUND MICROSCOPE
Around the beginning of the 1600's, through work attributed to the Janssen
brothers in the Netherlands and Galileo in Italy, the compound microscope
was developed.
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 observer's eye—with means of adjusting the position of
the specimen and the microscope lenses.

76. 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 more of how this is done
later).
For example, the total visual magnification using a 10x objective and a 15X
eyepiece is 150X I 7
Conversely, it may be (and often is) all too easy to degrade an image through
improper technique or poor equipment.
Essentially, this is how a microscope functions.
Light from a lamp passes through a sub stage condenser and then through a
transparent specimen placed over an opening in the stage.
Light is then gathered by the objective.

77. The objective, together with the built-in tube lens (more of this later),
focuses the image of the specimen at the level of the fixed diaphragm of
the eyepiece.
The image is then seen by the observer as if it were at a distance of
approximately 10 inches (250 millimeters) from the eye.
At the lowest part of the observation tube in infinity-corrected systems
for Olympus equipment, there is a tube lens which gathers the parallel
beams of light emerging from the objective and focuses the resulting
image at the plane of the fixed diaphragm of the eyepiece.
The eye lens of the eyepiece, together with the curved cornea and lens
of the eye, focuses the image on the retina of the observer's eye .

78. Binocular microscope

79. Light Source
• Light is an essential part of the system.
• Sunlight, low voltage electric lamps, tungsten etc.
• Neutral density filters- excess brightness of light
is reduced to comfortable viewing levels through
these filters.
The source of illumination should be
• Uniformly intense
• Should completely flood the back lens of the
condenser with light when the lamp iris
diaphragm is open
• Make the object appear as though it were self-
luminous

80. OBJECTIVE LENSES

81. Color Coding of Objective Lens
Immersion color code a Immersion type
Black Oil
Orange Glycerol
White Water
Red Special
Magnification color code b Magnification
Black 1x, 1.25x
Brown 2x, 2.5x
Red 4x, 5x
Yellow 10x
Green 16x, 20x
Turquoise blue 25x, 32x
Light blue 40x, 50x
Cobalt (dark) blue 60x, 63x
White (cream) 100x
a. Narrow colored ring located near the specimen of objective.
b. Narrow band located closer to the mounting thread than the immersion code.

82. THE MOST IMPORTANT OPTICAL COMPONENT OF THE MICROSCOPE
IS THE OBJECTIVE.
• Its basic function is to gather the light passing through the specimen and then to
project an accurate, real, inverted IMAGE of the specimen up into the body of the
microscope.
• Other related functions of the objective are to house special devices such as an iris
for darkfield microscopy, a correction collar for counteracting spherical aberration
(more of this later), or a phase plate for phase contrast microscopy.
• The objective must have the capacity to reconstitute the various points of the
specimen into the various corresponding points in the image, sometimes called the
"anti-points".

83. • The objective must be constructed so that it will be focused close enough to
the specimen so that it will project a magnified, real image up into the
microscope.
• The higher power objectives should have a retractable front lens housing to
protect the front lens where the objective requires focusing very close to the
specimen.
• To the extent possible, corrections for lens errors (aberrations) should be
made within the objective itself.

84. ACTION OF A CORRECTION COLLAR (DIAGRAMMATIC). SECOND
IMPORTANT OPTICAL COMPONENT IS THE EYEPIECE
• Its basic function is to "look at" the focused, magnified real image projected by the
objective (and tube lens in infinity-corrected systems) and magnify that image a
second time as a virtual image seen as if 10 inches from the eye.
• In recording, a Photoeyepiece "picks up" the real image projected by the objective a
second time as a real image able to be captured by a camera,
• The eyepiece houses a fixed diaphragm. It is at the plane of that fixed diaphragm
that the image projected by the objective will be "seen".
• On the shelf of the fixed diaphragm, the eyepiece can be fitted with scales or
markers or pointers or crosshairs that will be in simultaneous focus with the focused
image.

85. Eyepieces work in combination with microscope objectives to
further magnify the intermediate image so that specimen
details can be observed. Best results in microscopy require that
objectives be used in combination with eyepieces that are
appropriate to the correction and type of objective.
ABERRATION-FREE lox EYEPIECE WITH DIOPTER
ADJUSTMENT

86. There are two major types of eyepieces that are grouped according to lens and diaphragm
arrangement:
the negative eyepieces with an internal diaphragm and positive eyepieces that have a
diaphragm below the lenses of the eyepiece.
Negative eyepieces have two lenses: the upper lens, which is closest to the observer's eye,
is called the eye-lens and the lower lens (beneath the diaphragm) is often termed the field
lens.

87. In their simplest form, both lenses are piano-convex,
with convex sides facing- the specimen.
Approximately midway between these lenses there is a fixed circular opening or internal
diaphragm which, by its size, defines the circular field of view that is observed in looking
into the microscope.
RAMSDEN EYEPIECE, HLTYGENIAN EYEPIECE. BOTH
ILLUSTRATED IN LONGI- SECTION

88. SIMPLE EYEPIECE
The simplest kind of negative eyepiece, or Huygenian eye-
piece, is found on most routine microscopes fitted with achromatic objectives.
Although the Huygenian eye and field lenses are
corrected, their aberrations tend to cancel each other out.
not well
More highly corrected negative eyepieces have two or three lens
elements cemented and combined together to make the eye lens.
If an unknown eyepiece carries only the magnification inscribed
on the housing, it is most likely to be a Huygenian eyepiece, best
suited for use with achromatic objectives of 5x-40x magnification.
The other main kind of eyepiece is the positive eyepiece with a
diaphragm below its lenses, commonly known as the Ramsden
eyepiece.

89. • This eyepiece has an eye lens and field lens that are also piano-
convex, but the field lens is mounted with the curved surface
facing towards the eye lens.
• The front focal plane of this eyepiece lies just below the field
lens, at the level of the eyepiece diaphragm, making this
eyepiece readily adaptable for mounting graticules.
• To provide better correction, the two lenses of the Ramsden
eyepiece may be cemented together

90. THE THIRD IMPORTANT OPTICALCOMPONENT IS
THE SUBSTAGE CONDENSER.
1. Its basic function is to gather the light coming from the light source
and to concentrate that light in a collection of parallel beams (from
every azimuth) onto the specimen.
2. The light gathered by the condenser comes to a focus at the back focal
plane of the objective (later, the explanation of this term).
3. In appropriately set up illumination, it is arranged that the image of the
light source, comes to focus at the level of the built-in variable
aperture diaphragm of the substage condenser (the front focal plane of
the condenser).

91. 1. Correction for lens errors are incorporated in the finest condensers,
an important feature for research and photography.
2. Where desired, the condenser can be designed to house special
accessories for phase contrast or differential interference or dark field
microscopy.
The substage condenser is fitted below the stage of the microscope,
between the illumination lamp and the specimen. Condensers are
manufactured according to different levels of correction needed.

92. The simplest and least well-corrected condenser is the Abbe
condenser, numerical aperture up to 1.25. While the Abbe condenser is
capable of passing bright light, it is not well-corrected chromatically or
spherically.
As a result, the Abbe is most suitable for routine observation with
objectives of modest numerical aperture and correction.
A. ABBE AND APLANATIC-ACHROMATIC CONDENSER SYSTEMS.
B. CONES OF LIGHT TRANSMITTED

93. found in the aplanatic-achromatic condenser.
Such a condenser is well-corrected for
chromatic aberration and spherical aberration.
It is the condenser of choice for use in color
observation and recording in white light.
The engraving on the condenser includes its numerical aperture and its correction, if
aplanatic-achromatic. Condensers with a numerical aperture above 1.0 perform best
when a drop of oil is applied to their upper lens and is brought into contact with the
underside of the slide
CONE OF ILLUMINATION. THE SUBSTAGE CONDENSER MUST BE FOCUSED AND THE
DIAPHRAGM ADJUSTED SO THAT THE CONE OF ILLUMINATION COMPLETELY FILLS THE
APERTURE OF THE MICROSCOPE OBJECTIVE

94. • The condenser aperture and the proper focusing of the condenser are of critical
importance in realizing the full potential of the objective in use. Likewise, the
appropriate use of the adjustable aperture iris diaphragm (incorporated in the
condenser or just below it) is most important in securing excellent illumination and
contrast.
• The opening and closing of the aperture iris diaphragm controls the angle of the
illuminating rays which pass through the condenser, through the specimen and into
the objective.

95. • For low power objectives (4X or below), it may be necessary to unscrew the top
lens of the condenser or to use a condenser with a flip-top upper lens.
• Special low power condensers are also available.
• Specialty condensers are available for dark field microscopy, for phase contrast,
polarized light, and for interference microscopy.
• The height of the condenser is regulated by one or a pair of condenser knobs
which raise or lower the condenser.
• This adjustment is described in Koehler illumination

96. OTHER OPTICALCOMPONENTS:
1. The base of the microscope contains a COLLECTOR LENS. This lens is placed in
front of the light source. Its function is to project an image of the light source onto
the plane of the condenser's aperture diaphragm. In some instruments a diffusion or
frosted filter is placed just after the collector lens (side closer to the specimen) in
order to provide more even illumination.
2. Also in the base of the microscope, under the condenser, is a FIRST SURFACE
MIRROR (silvered on its front surface only). Its function is to reflect the light
coming from the lamp up into the sub stage condenser. Just before that mirror
(closer to the lamp side) is another variable diaphragm known as the field
diaphragm.

97. • At the lowest part of the observation tubes (binocular or trinocular) there is
incorporated a TUBE LENS.
• Its function is to gather the parallel rays of light projected by the objective (in
infinity-corrected systems) and bring those rays to focus at the plane of the fixed
diaphragm of the eyepiece. In the instruments of some manufacturers, the tube
lens is built into the body of the microscope itself.

98. MECHANICAL/ELECTRICAL COMPONENTS-
• The STAND of the microscope houses the mechanical/electrical parts of
the microscope. It provides a sturdy, vibration-resistant base for the various
attachments.
• The BASE of the Olympus microscopes is Y-shaped for great stability. It
houses the electrical components for operating and controlling the intensity of
the lamp. The lamp may be placed, depending on the instrument, at the lower
rear of the stand or directly under the condenser fitting. The base also houses
the variable field diaphragm. The base may also have built in filters and a
special circuit for illumination intensity for photomicrography

99. • Built into the stand is a fitting to receive the microscope STAGE. The stage has
an opening for passing the light. The specimen is placed on top of the stage and
held in place by a specimen holder. Attached to the stage are concentric X-Y
control knobs which move the specimen forward /back or left/right.
• On the lower right and left side of the stand are the concentric COARSE and
FINE FOCUSING KNOBS. These raise or lower the stage in larger / smaller
increments to bring the specimen into focus.

100. • Under the stage there is a built-in ring or a U-shaped CONDENSER HOLDER.
This holder receives any one of several types of condenser. The holder has a
tightening screw to hold the condenser in place and may have 2 small knobs (at 7
o'clock and 5 o'clock positions) for centering the condenser to the optical axis of the
microscope in Koehler Illumination (explained later). Adjacent to the condenser
holder there are either one or two knobs for raising or lowering the condenser.
• Above the stage, the stand has a NOSEPIECE (may be fixed or removable) for
holding the objectives of various magnifications. The rotation of the nosepiece can
bring any one of the attached objectives into the light path (optical axis). The
nosepiece may also have a slot forspecial attachments.

101. • Removable OBSERVATION TUBES, either binocular or trinocular, are attached to
the stand above the nosepiece.
• The binocular is used for viewing and the trinocular is used for viewing and /or
photography. The observation tubes are usually set at approximately a 30 degree
angle for comfortable viewing and may be tillable or telescoping push-pull for
greater flexibility.
• The bottom of the observation tube holds a special lens called the TUBE LENS.
• The tube lens has the function of gathering the parallel beams projected by the
objective and bringing the image to focus at the level of the eyepiece diaphragm
(intermediate image plane).
• On the instruments of some manufacturers (not Olympus) the tube lens may complete
optical corrections not made in their objectives.

102. THE MICROSCOPE STAND HAS THE FOLLOWING FUNCTIONS:
1. To insure stability and rigidity of the microscope.
2. To provide the frame for holding the objectives and eyepieces at opposite
ends of the stand.
3. To make it possible, by means of adjustment knobs, to bring the specimen into
focus.
4. To hold the specimen on a flat surface stage and to be able to move the specimen
on that surface.
5. To carry the moveable substage condenser which receives the light deflected by
the built-in mirror and transmits that light up through the specimen.
6. To hold the lamp and the electrical controls to operate the lamp and control its
brightness

103. ILLUMINATION
MICROSCOPE
ILLUMINATOR. THE
ESSENTIAL ELEMENTS
OF THE ILLUMINATOR
ARE THE LAMP, A
COLLECTOR LENS, AND
THE FIELD DIAPHRAGM.
THE DIAPHRAGM IS
ADJUSTABLE
• Since specimens rarely generate their own light, illumination is
usually furnished by means of a built-in lamp. The light beams pass
through the substage condenser after deflection by a built-in mirror.
• The light transmitted by the condenser then passes through the
specimen on the stage, into the objective, thus illuminating the
specimen.
If the lamp is of high intensity (tungstenhalogen), its
brightness is controlled by a built-in or separate
transformer.

104. TYPES OF ILLUMINATION:-
Critical or Nelsonion or source focused illumination.
• Homogenous light source is used to get this kind of
illumination.
• No lamp condenser used
• Normally employed with a base light source.
• Light source be focused on the object plane by racking
the substage condenser up and down.
• Light source must be large.
• Used in routine visual microscopy. Easy to set up.

105. KOHLER ILLUMINATION
• Non homogenous light source is used.
• Lamp condenser or collecter lens is essential.
• A Field iris is placed in front of the collecter to focus the light from the collecter
onto the substage iris.
• It is used with compound lamps .
• Used in photomicrography.
• Hence image obtained from the field iris will be found in the primary image plane
and retina of the eye.