The document provides an extensive overview of light microscopy, including its history and key figures such as Hans Janssen, Galileo, Robert Hooke, and Anton van Leeuwenhoek. It discusses the principles of light’s properties, refraction, reflection, and various types of lenses, as well as the components and classifications of light microscopes. Additionally, it covers concepts of magnification, resolution, numerical aperture, and the significance of various objective lenses in microscopy.

2. LIGHT MICROSCOPYY
• Dr SRUTHY P
• 3RD
MDS

3. INTRODUCTION
• Microscope, instrument that produces enlarged images of
small objects, allowing the observer an exceedingly close view
of minute structures.
• The microscope must accomplish three tasks:
produce a magnified image of the specimen
separate the details in the image
render the details visible to the human eye or camera

4. History…….
• 1590 - Hans & Zacharias Janssen of Middleburg,
Holland manufactured the first compound
microscopes
• While experimenting with several lenses in a tube,
they discovered a combination of two lenses in a
particular fashion.
• One lens was at the eyepiece of the microscope
(ocular), and the second lens was near the sample to
be studied (objective). This arrangement
significantly enlarges the sample under observation.

5. Galelio • In 1610, Galelio used a telescope at close range to
magnify the parts of insects
• 1624 invented a compound microscope
• During this time, the word "microscope" was beginning
to be accepted and regularly used by the members of the
"Academia dei Lincei," a scientific society that included
Galileo.
• Origin was from the Greek words μικρόν (micron) meaning
"small," and σκοπε ν
ῖ (skopein) meaning "to look at."

6. Robert Hooke
• Robert Hooke (1635-1703), is an English
scientist and mechanical genius, who was
educated at the University of Oxford.
• Hooke was also a pioneer in microscopic
research and published his observations, which
included the discovery of plant cells.
• His Micrographia was an important milestone in
proving the importance of microscopy, and
has granted him the title of English father of
microscopy
• Hooke also developed and modified the Jansen
compound microscope into a more modern
one, similar to the one we currently use.

7. Antoni van Leeuwenhoek
 A Dutchman, Anton van
Leeuwenhoek, is considered the father
of microscopes
 He worked as an apprentice in a dry
goods store where magnifying lenses
were used to count the threads in cloth.
 He made lenses which magnified up to
270x.
 This led to the first practical
microscopes.
 In 1674, Anton was the first to see and
describe bacteria, yeast, plants, and life
in a drop of water.

8. LIGHT AND IT’S PROPERTIES
Light or visible light, is electromagnetic radiation of a wavelength that is
visible to the human eye (about 400–700 nm)
Speed - approximate value of 3×108 meters per second
Travels in straight line
Illustrated as a sine wave

9. Wavelength – color , monochromatic or polychromatic
Amplitude – brightness, energy content of light

10. Refraction
Reflection
Dispersion

11. Refraction
• Bending of the light ray when
passing from one medium to
another is called refraction

12. The extent of refraction depends on
a) Angle of incidence
b) Density of medium
c) Curvature of interface between two medium
As a rule When light enters a less optically dense
medium it bends away from the perpendicular.
When light enters a more optically dense medium
it bends toward from the perpendicular.

13. • The angle to which light rays are deviated called as
angle of refraction .
• Ratio of sine values of angle of incidence (i) and
refraction (r) called as refractive index.

14. • It’s a dimensionless number that describes how fast light travels through
the material.
For example, the refractive index of water is 1.333, meaning that light
travels 1.333 times slower in water than in a vacuum.
• Increasing the refractive index corresponds to decreasing the speed of
light in the material.

15. • REFLECTION
• When a ray of light approaches a smooth
polished surface and the light ray bounces back, it
is called the reflection of light.
• The incident light ray which lands upon the
surface is said to be reflected off the surface.
• The ray that bounces back is called the reflected
ray. If a perpendicular were to be drawn on a
reflecting surface, it would be called normal. The
figure below shows the reflection of an incident
beam on a plane mirror.

16. Dispersion
• Separation of light into its constituent wavelengths when entering
a transparent medium - such as a prism
dispersion
• Short wavelengths are “bent” more than
long wavelengths

17. Lens:
• 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
Positive
Negative

21. • Parallel rays of light entering a simple
lens are brought together by refraction
to a single point, the principal focus or
focal point, where a clear image will be
formed of an object
• The distance between the optical
center of the lens and the principal
focus is the focal length
Focal
length
o

26. Real image formation in eye

27. Eg:magnifying lenses

28. a) Distant object
Real
Inverted
Smaller than object
At F
b) Object at 2F
Real
Inverted
Same size
At 2F
c) Object between 2F
and F
Real
Inverted
Larger than object
Beyond 2F
d) Object at F
No image
Refracted rays
are parallel
e) Object between F
and lens
Virtual
Erect
Larger than object
Behind the object on the

29. ABERRATIONS IN LENS SYSTEMS

30. Chromatic aberration
• Chromatic aberration, is a common optical problem that occurs when a lens
is unable to bring all wavelengths of color to the same focal plane.
• a “color fringing” or “purple fringing”,

31. Spherical aberration
is caused when the light rays entering a curved lens at its periphery are refracted more
than those rays entering the center of the lens and are thus not brought to a common
focus.

32. an achromatic doublet, composed of two individual lenses made from
glasses with different amounts of dispersion.
Typically, one element is a negative (concave) element made out of flint glass
such as F2, which has relatively high dispersion, and the other is a positive (
convex) element made of crown glass such as BK7, which has lower
dispersion.
The lens elements are mounted next to each other, often cemented together,
and shaped so that the chromatic aberration of one is counterbalanced by
that of the other.
achromat is a lens that is designed to limit the effects of chromatic and
spherical aberration

33. Classification Of Light Microscopes
Depending on lens system:
Simple And Compound
Depending on optical technique:
Bright Field, Dark Field, Phase Contrast, Interference
Depending on microscope frame type
Upright Microscope, Inverted Microscope

34. • One Light Path & One Lens = Simple Microscope
• One Light Path & Multiple Lenses = Compound Microscope

35. COMPONENTS OF A COMPOUND
MICROSCOPE

36. • 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

38. Light source
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
• Day Light
• Oil Lamp
• Low Voltage Electric Lamps
• 6V, 9W tungsten halogen lamp

39. Afocal or Nonfocused Illumination
• Illumination systems that do not form an image of the light source at some
point in the optical pathway are called afocal or nonfocused illumination.
• Before the invention of electric bulbs, microscopists were limited in their
choice of suitable sources for microscope illumination. During daylight hours,
they could point their microscopes (or substage reflector mirrors) towards the
sky.

40. Mirror (or internal lamp)
• Combined with an out board light
source the mirror serves to direct
light into the condenser.
• In most cases there are two
surfaces, a flat surface for
directing a parallel light beam into
the condenser, and a curved or
concave surface for directly
focusing light onto the specimen
with the condenser removed.

41. CONDENSER
• It is a vital part of the illumination system
Designed to collect, control and concentrate
light from the lamp on to the specimen.

42. Basically a group of lenses.
The substage condenser gathers light from the microscope light source and concentrates it
into a cone of light that illuminates the specimen with uniform intensity over the entire
view field.

43. • All condensers have an aperture
diaphragm with which the diameter of
cone of the light beam can be controlled.
• If the diaphragm is closed too much –
too contrast and refractile
• If the diaphragm is left wide open – glare
(due to extraneous light interference)
• In both cases – resolution - poor

44. • Each time an objective is
changed, a corresponding
adjustment must be performed
on the substage condenser
aperture iris diaphragm to
provide the proper light cone
for the numerical aperture of
the new objective.

45. Type of condenser
Abbe condenser •simplest, least corrected, least
expensive.
•2 lens elements.
Achromatic •4 lens elements
Aplanatic •5 lens elements
Aplanatic-achromatic •corrected for both chromatic
and spherical aberrations
•use in critical color
photomicrography with white
light;
• 8 internal lens

46. • The two-lens Abbe condenser is in
common use but is not very efficient,
forming only an imperfect image of the
light source.(NA = 0.25)

47. These are corrected chromatic
aberration .
NA – 0.95
Recommended for research microscopy
and for color photomicrography

48. • optically corrected for spherical
aberration
• NA =1.40

49. THE IRIS DIAPHRAGM
• 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.
• When the condenser aperture diaphragm is opened too wide, stray
light generated by refraction of oblique light rays from the
specimen can cause glare and lower the overall contrast.
• When the aperture is made too small, the illumination cone is
insufficient to provide adequate resolution and the image is
distorted due to refraction and diffraction from the specimen.
Correct setting
N.A of condenser is
matched to the N.A of
objective

52. • When properly adjusted, light from the condenser will fill the back
focal plane of the objective with image-forming light by projecting a
cone of light to illuminate the field of view .

53. OBJECT STAGE
 Rigid platform with an aperture through which
light pass.
 Allows movement of the object in two
directions by means of two micrometer threads.
• 3 x 1 inch slide
• Moves over an area approximately 3½ x 1¼
inches
• Vernier scale 0-80, 80-110.
• Useful to locate slide at same position at a
later date.

54. OBJECTIVE
• The most important lens of microscope
• collect maximum light possible from the object, unite it and form a high
quality real image
• Its properties largely determine depth of focus, resolution, and contrast of
the specimen.

55. • switching from one lens to another requires only a little turn of the fine
adjustment to bring the image into sharp focus.
• This arrangement of lenses is called parfocal system
• Parcentral : the image remains in the center of field of view when the lenses
are changed

56. TYPES OF OBJECTIVES
• Achromatic objectives
• Fluorite objectives
• Apochromatic objectives
• Plan objectives

57. Achromatic Objectives
• Chromatic aberration can be corrected to within useful limits by using a two
component lenses.
• A positive lens is combined with a negative lens made of glass producing a
greater chromatic aberration.
• The negative lens corrects the chromatic aberration in the positive lens, and
only partially neutralizes its magnifying power.
• This method will correct a thin positive lens for any two colors, leaving a small
error in the intermediate colors. This type of lens is known as an achromatic
lens.

58. Apochromatic Objectives
• They are highly corrected lens and always used for
photomicrography.
• They are corrected chromatically for three colors (red,
green, and blue),
• When fluorspar is incorporated in the glass of
achromatic lens, three colors can be brought to the
one focal point, and the amount of chromatic
aberration visible in the image will be negligible.

59. Fluorite Objectives (CaF2)
• Fluorite or semi apochromatic objectives have fluorite
incorporated into the lens system to give better color
correction. .
• They produce a quality image mid way between that of the
achromat and apochromat.

60. • Levels of optical correction for aberration in commercial
objectives.
(a) Achromatic objectives, the lowest level of correction,
contain two doublets and a single front lens;
(b) Fluorites or semiapochromatic objectives, a medium
level of correction, contain three doublets, a meniscus
lens, and a single front lens; and
(c) Apochromatic objectives, the highest level of correction,
contain a triplet, two doublets, a meniscus lens, and a
single hemispherical front lens.

61. Plan Objectives
• All three types of objectives suffer from pronounced field curvature and project
images that are curved rather than flat.
• To overcome this inherent condition, lens designers have produced flat-field
corrected objectives that yield flat images. Such lenses are called plan achromats,
plan fluorites, or plan apochromats.
• In Plan Objectives, when imaging the field, parts of the specimen at the edge of
the visual field are almost as well focused as those in the center.
• A Plan Objective lens will also have the same corrections for spherical and
chromatic aberrations .

62. • Polarizing objectives - strain-free objectives for use on the polarizing
microscope
• Phase objectives contain a phase-plate for use in phase-contrast microscopy

63. • Every objective has a fixed
working distance
focal length
magnification and
numerical aperture(NA).

65. Magnification
• Total magnification of microscope =
magnification of objective x magnification of eye
piece.
• With the eye piece of 10x, the total
magnifications with the three objectives will be as
follows

66. • Total magnification is the product of magnification values of the objective and
eyepiece, provided the system is standardized to an optical tube length of 160mm
• For variation
M= optical tube length x M of eyepiece
focal length of O
• With lower magnification, a large area on the slide can be seen, with higher
magnification, though details can be seen, the area of view is restricted.

67. • Useful magnification range - At a selected numerical aperture, when the
microscope provides a magnified image that has a magnitude equal to the
resolution limit of the human eye and any additional magnification beyond this
point does not result in the resolution of even smaller specimen detail
• Microscope resolution is limited by NA and wavelength
• Empty magnification: Increase in size of image which does not increase
information. Magnification which does not contribute to improved resolution or
resolving power and which is beyond what the system can deliver.

68. Resolution
• The ability of a lens to define detail.
• ie the ability to tell two points apart as separate points. If the resolving
power of your lens is 2um that means two points that are 2um apart can
be seen as separate points. If they are closer together than that, they will
blend together into one point.
• Resolution is restricted by two factors: (1) the numerical aperture of the
lens; and (2) the wavelength of light employed. The relationship is as
follows
• Resolution=1.2 lambda/2 NA
• Lambda –wavelength of light employed

69. Numerical aperture
• the numerical aperture (NA) of an optical system
is a dimensionless number that characterizes the
range of angles over which the system can accept
or emit light.
• Numerical aperture of objective lens is the ability
of the objective lens to gather the cone of light
from the specime.
• higher the NA the greater the resolution
NA = n sin µ(n=the refractive
index of the medium between the object and
the lens)
µ is half of the angular aperture(maximum cone of light) of the objective
The apertures are measured by, the angle formed by the outer edges of the lens, and a point
on the object.

71. Condenser
Object
Real image
Eye lens
Eye piece lens
Objective
Virtual image

72. Refractive Index • Higher numerical apertures can be obtained by
increasing the imaging medium refractive index
(n) between the specimen and the objective
front lens.
• Microscope objectives are now available that
allow imaging in alternative media such as water
(refractive index = 1.33), glycerin (refractive
index = 1.47), and immersion oil (refractive
index = 1.51).

73. Color-Coded Rings on Microscope Objectives
• Immersion color code
• Black - Oil immersion
• Orange - Glycerol
immersion
• White - Water immersion
Magnification color code
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

78. NOSEPIECE: • Nose piece is fitted at the lower end of the body
tube and a number of objectives (usually three) are
fitted on it
• The nosepiece should bring each objective into its
central position. Any lens can be rotated into
position when desired, its correct position being
indicated by a ‘click’
• 18 mm

79. BODY TUBE
• Supports the eyepiece and objectives
• Standard 160 mm = 142 + 18 mm
• connects the eyepiece to the nosepiece
Monocular
Binocular
A)Seidentopf (sigh-den-top-ff) binocular head
B)Slider binocular head
Combined photo binocular

80. • Mechanical Tube Length is the
distance between the nosepiece to the
top edge of the observation tubes
where the eyepieces (oculars) are
inserted

82. Monocular microscopes
• Microscopes with one body tube
© J.Paul Robinson

83. Binocular microscopes
• The light rays emerging from the objective in the binocular
microscope are equally divided between the two eyepieces.
• The modern binocular microscope, achieves this by the use of
four prisms thus eyes will receive two parallel beam of light.
• Advantages
• Long period may be spent viewing through with minimum
amount of eye fatigue
• The eye pieces can be easily moved together or apart to adjust
inter ocular distance to suit individual requirements.

84. Types
A)Seidentopf (sigh-den-top-ff) binocular head
• A head design where the increasing or decreasing the
distance between the two eyepieces (interpupillary
distance) is done by twisting the eyepieces in an up and
down arc motion similar to most binoculars.
B)Slider binocular head
• Inter-pupillary distance is adjusted side-to-side, by sliding
the eyepieces towards and away from each other.

85. Photo binocular
• It has a prism system allowing
100% of the light to go either to
the observation eye piece, or to
camera located on the vertical
part, and some times has a beam
splitting prism dividing the light

86. Two body tube styles are also available;
• Dual head: has one vertical eyepiece lens and a second eyepiece
off the side at 45 degrees (so that two people can view the
sample at one time, or one person and camera).
• Trinocular: a microscope with a vertical tube at the top and
regular binocular eyepieces at 30 degrees. Vertical tube is often
used for a digital camera or a second observer.

87. Light pathway in binocular
The light rays emerging from the objective in the
binocular microscope are equally divided between
the two eyepieces.
It is not sufficient simply to insert a single prism
and divert one half of the rays, since this would
cause eyestrain due to both the observer's eyes
being focused on a single point a short distance
away, and the advantage of a binocular
microscope is that long periods may be spent
viewing through it with the minimum amount of
eye fatigue.

88. • The modern binocular microscope achieves
this by the use of four prisms.
• from Figure the eyes are receiving two
parallel beams of light.
• The lower central prism consists of two
prisms cemented together, at the interface
of which there is a semi-silvered surface :
this silvering is a very special process, fine
grains of silver being deposited so that
alternate light rays are differentially treated,
one being reflected to the right (Figure )
and the other passing into the upper prism.

89. • The light rays passing through the semi-silvered surface to the upper prism
travel through a greater thickness of glass than those that are reflected —
having the effect of retarding them — and this is compensated for by
making the right-hand prism with an extra thickness of glass as will be seen
by comparing the two outside prisms in Figure 30.15.

90. Finite and Infinite body tubes
• Over the past 10 years, the major microscope manufacturers have largely all
migrated to the utilization of infinity-corrected optical systems in both
research-grade biomedical and industrial microscopes.
• In these systems, the image distance is set to infinity, and a tube (or telan) lens
is strategically placed within the body tube between the objective and the
eyepieces (oculars)
• Here objective produces a flux of parallel light wave trains imaged at infinity
(often referred to as infinity space)which are brought into focus at the
intermediate image plane by the tube lens

92. EYE PIECE
• The final stage in the optical path of the
microscope
• function is to magnify the image formed by the
objective with in the body tube, and present the
eye with a virtual image.
• 5x, 8x, 10x eyepieces
• Two lenses-one at the top-the eye lens and the
other ‘field lens’ fitted at the bottom.

93. • Approximately mid-way 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.
• Eyepieces can be adapted for measurement purposes by adding
a small circular disk-shaped glass reticle (graticule or reticule) at
the plane of the field diaphragm of the eyepiece.
• Reticles usually have markings, such as a measuring rule or grid,
etched onto the surface. Because the reticle lies in the same
plane as the field diaphragm, it appears in sharp focus
superimposed over the image of the specimen.

94. Negative eyepieces
• In Negative eyepiece the focus is within (between) the lenses
of the eyepiece.
• The lower or field lens collects the image that would have
been formed by the objective and cones it down to a slightly
smaller image at the level of the field stop with in the
eyepiece, the upper lens then, produces an enlarged virtual
image which is seen by the microscopist.
• In their simplest form, both lenses are plano-convex, with
convex sides facing the specimen.

95. Positive eyepieces
• In Positive eyepieces the focus is outside the eyepiece lens system.
• The field stop (or diaphragm) is outside the eye piece, from which
the virtual image is focused and magnified by the entire 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. The front focal plane of this eyepiece
lies just below the field lens, at the level of the eyepiece fixed
diaphragm

96. Huygenian & Ramsden Eyepiece
• Negative oculars
• Originally designed for telescopes
• Most commonly used in microscope
• Positive oculars
• Compensated eye pieces are mostly of
the Ramsden type
Huygenian Eyepiece
Ramdsen Eyepiece

97. Kellner or Achromat Eyepieces
• Also called an "achromatized Ramsden".
• Kellner eyepieces are a 3-lens design. An
achromatic doublet is used in place of the
eye lens in the Ramsden design to correct
the residual chromatic aberration.
• They are inexpensive and have fairly good
image from low to medium power and are
far superior to Huygenian or Ramsden
design.

98. High-eye point oculars
• With normal eyepieces the distance between the top of the eyepieces and the ‘exit pupil’
(eye point) is so small as to prevent the wearing of glasses
• Usually eyes are placed 8-10 mm above the eye lens.
• High point oculars have viewing distances approaching 20- 25 mm above the surface of the
eye lens.
• Today, high eyepoint eyepieces are very popular, even with people who do not wear
eyeglasses, because the large eye clearance reduces fatigue and makes viewing images
through the microscope much more comfortable.

99. Compensating eyepieces
• Simple eyepieces such as the Huygenian and Ramsden will not correct for residual
chromatic difference of magnification in the intermediate image
• To remedy this in finite microscopy systems, manufacturers produce compensating
eyepieces that introduce an equal, but opposite, chromatic error in the objective lens
elements.
• Compensating eyepieces may be either of the positive or negative type, and can be
used at all magnifications with fluorite, apochromatic and all variations of plan
objectives

100. SUPPORT SYSTEM
• FOOT
• LIMB
• FOCUSSING SYSTEM

101. FOOT (BASE)
• It rests on the bench top and supports the stage and body of the microscope,
and in many cases also houses the lamp

102. LIMB (ARM)
• The arm is attached to the foot and supports the body tube

103. FOCUSSING SYSTEM
• Consists of coarse and fine adjustment screw-heads.
• It is employed for raising or lowering the optical system with reference to
the slide under study till it comes into focus
• Coarse- rack and pinion
• Fine adjustment- micrometer screws

104. OPERATION OF
MICROSCOPES

105. Condenser
Object
Real image
Eye lens
Eye piece lens
Objective
Virtual image

106. There have been varying approaches to maximize the
illumination within the microscope.
Critical illumination is used in simple equipment where
the light source is focused by the substage condenser in
the same plane as the object, but this produces uneven
illumination.
Köhler illumination is used for photography and more
specialized microscopes where an image of the light
source is focused by the lamp collector or field lens in the
focal plane of the stage condenser on the aperture
diaphragm.
The image of the field or lamp diaphragm is focused in
the object plane and the illumination is even (Fig. 3.13).
The illumination must be centered with respect to the
optical axis of the microscope to prevent poor resolution
CRITICAL ILLUMINATION(NELSONS) ILLUMINATION AND
KOHLERS ILLUMINATION

107. Illuminating apparatus
• Light emitted from the tungsten-halogen
lamp filament first passes through the
collector lens located close to the lamp
housing, and then through a second lens
that is closer to the field diaphragm.
• The second lens in the light path is called
the field lens, which is responsible for
bringing the image of the filament into
focus at the plane of the aperture
diaphragm.

108. • Focused light leaving the field lens is
reflected by a mirror (positioned at a 45-
degree angle to the light path) through the
field diaphragm and into the substage
condenser.
• The field diaphragm serves as a virtual
source of light for the microscope and its
image is focused by the condenser onto the
specimen plane

109. • The field diaphragm in the base of the microscope controls only the width of the bundle
of light rays reaching the condenser
• It does not affect the optical resolution, numerical aperture, or the intensity of
illumination.
• Proper adjustment of the field diaphragm is important for preventing glare than can
reduce contrast in the observed image.
• When the field diaphragm is opened too far, scattered light originating from the specimen
and light reflected at oblique angles from optical surfaces can act to degrade image quality.

110. Closing or opening the condenser diaphragm controls the angle of the light rays emerging
from the condenser and reaching the specimen from all azimuths.
Because the light source is not focused at the level of the specimen, the light at specimen
level is essentially grainless and extended and does not suffer deterioration from dust and
imperfections on the glass surfaces of the condenser.
Opening and closing of the condenser aperture diaphragm controls the angle of the light
cone reaching the specimen.
.

111. • Parallel light rays that pass through and illuminate the specimen are
brought to focus at the back focal plane of the objective, where the
image of the variable condenser aperture diaphragm and the image of
the light source will be seen in focus.

112. Field of view
• The diameter of the view field in an optical
microscope is termed the field number and represents
the diameter of the field measured in millimeters at the
intermediate image plane
• From this can be calculated the actual diameter of the
specimen being viewed
Field Size = Field Number (fn) ÷ Objective Magnification (Mo)

113. HOW TO OBSERVE A SLIDE ?
113

114. CAUSES OF ERROR IN FOCUSING
• Revolving Nose Piece is off centre
• Preparation is upside down
• Thick cover slip
• Dirt or Dried oil over Lens
• Air bubble in immersion oil
• Poor illumination – Condenser not fully racked up

115. MICROMETRY
• The standard unit of measurement in microscopy is a micrometer
(m), which is 0.001mm.
• The stage micrometer - 3 x 1 inch slide on which a millimeter scale
is engraved in 1/10 and 1/100 graduations.
• Eyepiece micrometer- disc with an arbitrary scale on it.
huygenian – placed on field stop
kellner - disc permanently in place

116. PROTOCOL FOR THE USE OF MICROSCOPE
• Step1: Examine the slide with naked eye
• Step 2: Slide is viewed under low magnification to get a general view all
over. Lower the stage about 7-8 cm below the body tube. Put slide on the
stage and bring the specimen over the central aperture.
Looking from the side, and using the coarse adjustment, bring the stage
up, so that lens is about 1 cm above the slide. Now look into the eyepiece
and adjust till the specimen comes into focus. Scan the entire field,
racking the fine adjustment all the time.

117. • Step 3: Choose an area of interest for viewing under high magnification and
focus under high power objective.

118. Focusing under oil-immersion objective
 most frequently used in hematology because of its high magnification
and resolution.
 Two important feature of this lens are its very small aperture and
deep focusing position, which is very close to the slide because of its
short focal length of about 2mm.
 cedar wood oil or Medias like glycerin or liquid paraffin during use.

119.  Raise the body tube so that the lens is about 8-10 cm above the slide. Place a drop of cedar
wood oil on the slide, and looking from the side, slowly bring the objective down till it just
enters the oil drop. The oil will spread out in the capillary space between the slide and the lens.
While looking into the eyepiece raise the objective with coarse adjustment till the cells come
and into view.
 After use, clean the objective with xylene and then with alcohol.

120. Care of the microscope
• Apart from cleaning the outer surface of their lenses, objectives are best left alone.
• Prisms should never be touched, and cleaning should be confined to blowing of the dust
• Lenses should be wiped only with fresh lens tissue or cotton wool, otherwise they may be
scratched.
• Immersion oil should be removed with lens tissue or cotton wool damped with xylene,
or, preferably ether

121. • Hold a microscope firmly by the stand, only. Never grab it by the eyepiece
holder, for example
• Hold the plug (not the cable) when unplugging the illuminator
• Since bulbs are expensive, and have a limited life, turn the illuminator off
when you are done

122. Cleaning and maintenance
Daily cleaning routine:
• The microscope should be dusted daily
• outer surface of the lenses of objectives polished with lens tissue or cotton wool.
• The top lens of the eyepiece should be polished to remove dust or fingermarks
• the microscope is set up for correct illumination.
• The substage condenser and the mirror should be cleaned.

123. Weekly cleaning routine
• The slides of the coarse adjustment, the mechanical stage and the substage
condenser should be wiped with a cloth dampened with xylene to remove dust
which would otherwise damage the slides.
• A little oil (as supplied for lubricating microscopes) is applied and the slides
replaced: later models do not require this treatment.
• The lens system should be checked and cleaned

124. Limitations of standard optical microscopy
(bright field microscopy)
• The technique can only image dark or strongly refracting objects effectively.
• Diffraction limits resolution to approximately 0.2 micrometre.
• Out of focus light from points outside the focal plane reduces image clarity.
• Live cells and internal structures of the cell could not be studied effectively.
• Live cells lack sufficient contrast to be studied successfully, internal structures of the
cell are colourless and transparent. So to increase contrast special stains and dyes were
used but this caused killing and fixing the cells. Staining may also produce artefacts.

125. CONCLUSION
• The light microscope, often the symbol of research and scientific discovery,
has evolved over the last 350 years from Antonie van Leeuwenhoek’s simple
magnifier to the more sophisticated instruments of today.
• Studies of biological structures and processes on both fixed and live
specimens have advanced light microscopy into an indispensable tool for cell
and molecular biologists.

126. REFERENCES
• Theory and practical of histological techniques: John D Bancroft
• Cellular pathology technique: 4th
edition by C.F.A Culling, R.T.Allison & W.T
Barr
• Basics in Light Microscopy- Dr. Arne Seitz, PT-BIOP Course, 2010, EPFL