LIGHT MICROSCOPE

1. LIGHT MICROSCOPE
BY-Dr.Anubhuti Mohania

2. REFERENCES
• Praful B.Godkar,Darshan P.Godkar,Textbook of Medical
Laboratory Technology,Second Edition.
• John D. Bancroft,Theory and Practice of Histological
Techniques,Fifth Edition.
• C.F.A Culling,Handbook of Histopathological and
Histochemical Techniques,Third Edition.
• Dr.V.G.Ranade,Laboratory Manual & Journal of
Physiology.
• Prof.A.K.Jain,Manual of Practical Physiology for BDS.
• A.I.T.B.S. publishers India,Manual of Basic Techniques for
a Healthy Laboratory,Second Edition.
• Prescott,Harley,Klein,Microbiology,Fifth Edition.

3. CONTENTS
• Introduction
• History of Microscope
• Classification of Microscope
• Lens & its Defects
• Light & its Properties
• Components of Microscope
• Types of Light Microscope
• Cleaning of Microscope

4. INTRODUCTION
• MICROSCOPE is an optical instrument used to visualise
very small objects.
• A simple microscope has only one lens,whereas a compound
microscope has a set of lens,which magnify the image.The
image formed by the first lens acts as the object for the
second lens.So the advantages are that the magnification &
resolution is higher in a compound microscope.

5. HISTORY OF MICROSCOPE
• The early simple “microscopes”
which were really only
magnifying glasses had power,
usually about 6X - 10X .
• These early magnifiers were
called “flea glasses”. Flea
glasses were small simple
microscopes used to study
insects (and other small
objects) .

6. • Sometime around 1590, two Dutch
spectacle makers, Zaccharias Janssen
and his father Hans put several
lenses in a tube and made a very
important discovery. The object near
the end of the tube appeared to be
greatly enlarged, much larger than
any simple magnifying glass could
achieve by itself.They had invented
the compound microscope.
Zaccharias Janssen

7. • Galileo described the
principles of lenses and light
rays and improved both the
microscope and telescope.
Galileo

8. • Anthony Leeuwenhoek of Holland while
working in a dry goods store used the
magnifying glass to count threads in woven
cloth.
• He saw bacteria, yeast, blood cells and many
tiny animals swimming about in a drop of
water. Anthony Leeuwenhoek has been called
the "Father of Microscopy".
• Using microscopes, he was the first to observe
and describe single-celled organisms, which he
originally referred to as animalcules, and
which we now refer to as microorganisms.
• He was also the first to record microscopic
observations of muscle fibers, bacteria,
spermatozoa. It was he who discovered
bacteria, free-living and parasitic microscopic
protists, sperm cells, blood cells, microscopic
nematodes

9. • Robert Hooke, an Englishman
sometimes called the “English
Father of Microscopy”. Robert
Hooke wrote Micrographia,
the first book describing
observations made through a
microscope. Hooke was the
first person to use the word
"cell" to identify microscopic
structures when he was
describing cork.

10. TYPES OF MICROSCOPE
1. Light microscope
2. Electron microscope
3. Probe microscope
BASED ON NUMBER OF LENS
1. Simple microscope
2. Compound microscope
BASED ON ILLUMINATION
1. Bright field microscope
2. Dark field microscope
3. Phase contrast microscope
4. Interference microscope
5. Polarising microscope
6. Florescence microscope

11. CLASSIFICATION OF MICROSCOPES :
(1) Magnetic field based
(2) Electric Field based
(3) Electromagnetic field base
a) Atom beam
b) Electron beam
c) Deep ultraviolet (230 nm-350 nm)
d) Infra red
e) Visible light
Continuous light
Discrete light Phosphorescence
Fluoroscence
f) Radio Frequency
g) Microwave
h) X-ray
i) Gamma ray
j) others
MICROSCOPES BASED ON ELCTRON BEAM
Transmission Electron Microscope
Scanning Electron Microscope
Scanning Transmission Electron Microscope

12. LENS
• Lens –name given to a piece of glass or other transparent
material usually circular ,having two surfaces ground & polished
in a specific form in order that rays of light passing through it
shall either converge(collect together) or diverge(separate).

13. • A lens is called positive when
it causes light rays to
concentrate or converge to
form real image.
• A lens is called negative when
light rays passing through will
diverge or scatter & positive
or real images not seen.
• Positive lenses-thicker at
centre than periphery.
• Negative lenses-thinner at
centre.

14. Defects of a lens
• Chromatic aberration
• Spherical aberration
CHROMATIC ABERRATION-
o white light is composed of all
spectral colors & on passing
through a simple lens-each
wavelength will be refracted to a
different extent,blue being brought
to a shorter focus than red.

15. o It results in unsharp image
with colored fringes.
o To correct this it is possible to
construct compound lenses of
different glass elements.
 An ACHROMAT is corrected for
two colors blue and
red,producing a secondary
spectrum of yellow/green which
in turn can be corrected by
adding more lens components-
APOCHROMAT.

16. • SPHERICAL ABERRATION-
o It is caused when light rays
entering a curved lens at its
periphery are refracted more
than those rays entering the
center of lens and thus not
brought to a common focus.
o These faults can be corrected
by making combinations of
lens elements of different
glass like fluorite & of
differing shapes.

17. LIGHT & ITS PROPERTIES
• Light radiates in all directions
from its source.
• Unless something interferes,it
travels in a straight line to
infinity.
• The passage of light in optics of
microscope ,ray & bundle of
rays are drawn as straight lines
but to demonstrate theory that
light is a form of energy as a
series of pulses from its source
–it is shown as a sine curve
representing waves of energy

18. • AMPLITUDE-refers to strength of
energy or brightness of light.
• WAVELENGTH-distance between
apex of one wave and the
next,measured in nanometers.
• FREQUENCY-number of waves per
second ,remains constant.
• COHERENT-individual rays of
identical frequency from the same
source.
• NON COHERENT-rays from
different sources or of different
frequencies.

19. • RETARDATION &
REFRACTION
• Media through which light is
able to pass will slow down or
retard the speed of light in
proportion to density of
medium
• Higher the density-greater
the degree of retardation.

20. • Rays of light entering a sheet
of glass at right angles are
retarded in speed but their
direction is unchanged.
• If light enters the glass at any
other angle,deviation of
direction will occur in
addition to retardation-called
REFRACTION.
• A curved lens –exhibits both
RETARDATION &
REFRACTION.

21. • The angle to which the rays are
deviated within the glass or other
transparent medium –called ANGLE
OF REFRACTION.
• Ratio of sine values of angles of
incidence & refraction-gives
REFRACTIVE INDEX of medium.
• Greater the refractive index-higher
the density of medium.
• Air -1.00
• Water-1.30
• Glass-1.5

22. • Light passing from rarer to
denser medium-refracted
towards normal & light
passing from denser to rarer
medium-refracted away
from normal.
• Angle of incidence may
increase to a point where
light emerges parallel to
surface of lens.beyond this
angle of incidence ,TOTAL
INTERNAL REFLECTION will
occur & no light will pass
through.

23. • IMAGE FORMATION-
• Parallel rays of light entering a simple lens
are brought together by refraction to a single
point,PRINCIPAL FOCUS or FOCAL
POINT,where a clear image will be formed of
an object.
• distance between optical centre of lens and
principal focus –FOCAL LENGTH.
• A lens also has other pairs of points one on
either side of lens called CONJUGATE FOCI.
(object placed at one will form a clear image on
screen placed at other).
• As the object is moved nearer the lens image
will be formed further away at greater
magnification and inverted.this is REAL
IMAGE-formed by objective lens of
microscope.

24. • If object is placed yet nearer
the lens,within the principal
focus,image is formed on
same side as object,is
enlarged right way up and
cannot be projected on a
screen-VIRTUAL IMAGE.and
is formed by eyepiece of
microscope of the real image
projected from objective.
• This appears to be at a
distance of approx. 25 cm
from the eye-around the
object stage level.

26. COMPONENTS OF MICROSCOPE
• LIGHT SOURCE/ILLUMINATION-
Daylight-inconvenient owing to its inconstancy.
Most commonly employed-electric filament lamp.
Lamp may either be a simple pearl bulb or a high intensity lamp.
SOURCE OF ILLUMINATION SHOULD BE-
o Uniformly intense
o Should completely flood the back lens of condenser with light
when the lamp iris diaphragm is open.
o Make the object appear as though it were self luminous.

27. 1. UNIFORM INTENSITY OF ILLUMINATION-60 watt pearl bulb is
used.KOHLER ILLUMINATION may be used.
2. The source of light should be sufficient to enable its rays when
directed by plane side of mirror to flood the back lens of
condenser uniformly.The high intensity type of lamp has an
optical axis & must be correctly aligned for use,& the distance
from the microscope at which it is adjusted so that lens
magnifies lamp image to correct size,a built in light source has
been so adjusted.

28. 3. The object will behave as if self luminous if the bulb or the image
of the lamp condenser is focussed in the object plane with the sub
stage condenser.

29. • 2 universally recognised methods for correct illumination-
NELSON METHOD-for this method the light source should be
homogenous and no lamp condensers used.
KOHLER METHOD-light source does not have to be homogenous,but
a lamp condenser is essential to project an image of the lamp
filament on to the substage iris diaphragm.In this system the
lamp condensing lens (evenly illuminated) functions as a light
source.

30. CRITICAL ILLUMINATION-Light source is focused by substage condenser in
same plane as object is in focus
KOHLER ILLUMINATION-an image of light source is focused by lamp
collector or field lens in focal plane of substage condenser.the image of the
field or lamp diaphragm will now be focused in object plane & illumination
is even.The image of light source & aperture diaphragm will in turn be
focused at the back focal plane of objective & can be examined with
eyepiece.

31. • CONDENSERS.
• Light from lamp-directed into
substage condenser(directly or by
mirror or prism).
• Purpose is to focus/concentrate
available light into plane of
object.
• Are provided with adjustment
screws for centering light path.
• All condensers have an aperture
diaphragm with which the
diameter of light beam can be
controlled.
• The condenser should form a
perfect image of the light source
and have the same numerical
aperture as the objective with
which it is being used.(achieved
approx. by removing eyepiece
,viewing substage iris diaphragm
in back focal plane of objective &
closing it down to two thirds of
field view).

32. • Adjustment of iris
diaphragm will alter size &
volume of cone of light
focused on object.
• If diaphragm is closed too
much image –too contrasty
& refractile.
• If diaphragm is left wide
open image will suffer from
glare (light interference)

33. o The two lens Abbe condenser is in common use,not very
efficient,forming imperfect image of light source.(should not be
used with apochromatic or fluorite lenses).
o A three lens aplanatic or a more highly corrected achromatic
condenser will give a good image with good resolution.(usually
fitted with swing out front lens,unscrewed,to illuminate whole
field for low power lenses.by swinging out front lens NA of
condenser is reduced to 0.3-0.4. for critical microscopy with
objectives having an NA exceeding 1.0 ,immersion oil should be
applied between condenser & slide,as well between objective &
slide.

34. COMPONENTS OF MICROSCOPE
• MECHANICAL PARTS
 BASE-U or V shaped metallic
structure used for keeping the
microscope stable.
 ARM OR HANDLE-metallic structure
used for holding the microscope.
 BODY TUBE-
o wide metallic tube which holds the
image draw tube.
o connects eyepiece to objective and
distance from objective to eyepiece is
called MECHANICAL TUBE LENGTH.
o 160 – 170 mm.
o the depth of nosepiece will affect the
tube length & its generally 18 mm in
depth,the actual length of body tube is
142 mm.

35.  DRAW TUBE-narrow metallic tube.the eyepiece is connected
at its upper end & nose piece at its lower end.
 NOSE TUBE-circular metallic revolving plate on which the
objectives are fitted in.
 STAGE-square or rectangular plate with a hole in centre on
which the slide is placed .There are clips to hold the
slide.There is also arrangement for movement of the slide in
horizontal or vertical direction.It also has a vernier calliper.

36. • OBJECTIVES ARE-
1. LOW POWER OBJECTIVE-10 x;NA=0.25
This lens magnifies the image 10 times .
Provides maximum field of view.
2. HIGH POWER OBJECTIVE-40x ;NA=0.65
Magnifies the image 40 times.
3. OIL IMMERSION OBJECTIVE-100x ;NA=1.30
Magnifies image 100 times.
4. SCANNING OBJECTIVE- very low power objective.
Allows viewing or scanning of much larger area on slide.
• COLOR CODES FOR OBJECTIVE
• RED-4x
• YELLOW-10x
• BLUE -40x
• BLACK-100x

37. • The objectives are often so constructed that when one lens is in
focus,the other lenses are more or less in focus.thus switching over
from one lens to another requires only a turn or two of the fine
adjustment screw to bring the object into sharp focus.this
arrangement of lenses is called PARFOCAL system.

38. • OBJECTIVE LENSES-collects maximum light from object .
• Fitted in revolving nosepiece.
• Slight pressure on lens when it touches a slide pushes it into its
body preventing damage to slide or lens.
• Magnifying power & numerical aperture(NA) of each lens is
etched on it.
• every objective lens has affixed working distance ,focal
length,magnification,numerical aperture.
• WORKING DISTANCE-it is the distance between object in focus &
front of lens system of object.
• FOCAL LENGTH-it is the distance from the centre of the simple
lens to a point at which the parallel rays are brought to sharp
focus.in a compound lens it is the distance between the object in
focus & a point approx. half way between component lenses of
objective.

39. • Within the objective there may be lenses and elements from 5 to
15 in no.
• NUMERICAL APERTURE-ability of lens to distinguish surface
details in specimen known as resolving power expressed in terms
of numerical aperture.
• RESOLUTION
X=1.2λ
2 NA

40. • Ability of an objective to resolve
detail is indicated by its NA.
Angle of cone of light from point source of object actually entering lens
when dry is only 78 degrees compared with 120 degrees in oil immersion
lens.

41. • EFFECTS OF HIGH NUMERICAL APERTURE
High NA increases resolution of objective-disadvantages are-
o It reduces depth of focus i.e ability to focus on more than one
layer of an object at same time.
o It reduces the flatness of field ,so that the edges are out of focus.

42. • Types of objectives
1. Achromats
2. Apochromats
3. Plane apochromats
4. Plane achromats
5. Fluorite objectives-have fluorite incorporated .they are corrected
for three wavelengths of light in yellow green of spectrum and
free of color fringes.
6. Polarising objectives-these are stain free objectives in polarising
microscope.
7. Phase objectives-contain a phase plate for use in phase contrast
microscopy.

43. • MAGNIFICATION-it is a factor of magnification values of eye
piece & objective.
• Magnification in a standard microscope with tube length of
160mm is calculated as
• Magnification=tube length
focal length
of objective

44. • For microscope with tube length other than 160 is calculated as
Magnification=tube length×eyepiece magnification
focal length of objective

45. • EYE PIECE-to enlarge the primary
image formed by objective.percieved
by eyes as virtual image.
• Optical distance is 250mm from eye
• Types
• -negative-focus is within(between)
lenses of eyepiece.it is composed of
two lenses –the lower or field lens
collects image formed by objective &
cones it down to a slightly smaller
image at level of field diaphragm
within eyepiece.the upper lens then
produces enlarged virtual image seen
by microscopist.

46. • - positive-focus is outside the
eyepiece lens system.It may
be used as a simple
microscope.the diaphragm is
outside the eyepiece from
which virtual image from
objective is focused &
magnified by entire eyepiece.

47. Types
1. Ramsden Eyepieces-positive oculars
2. Huygeinian Eyepieces-designed by HUYGENS for
telescope.negative oculars
3. Compensating-intended for use with apochromatic
objectives only.
4. Wide field-give a large flat field of view.
5. High eyepoint-for spectacle wearers, with normal
eyepieces distance between top of eyepiece & exit pupil
is so small –prevents wearing of glasses,made possible
through these eyepieces.

48. • ADJUSTMENT KNOBS
1. Coarse
2. Fine
Paired,2 on each side.
With the help of these adjustments height of the tube can be
adjusted insuch a way so that objective lens can be positioned at
its optimal working distance(its focal length) from object.the
fine adjustment is usually graduated in 1/50 ths & each
division corresponds to a movement of 0.002 mm of tube.It is
used for accurate focussing.

49. TYPES OF LIGHT MICROSCOPE
• Bright ground microscope
• Phase contrast microscope
• Interference microscope
• Polarising microscope
• Florescence microscope

50. DARK GROUND MICROSCOPE
• Staining aids the formation of images by absorbing part of the
light(some wavelengths) & producing an image of amplitude
differences & color.
• unstained sections have refractive indices close to that of medium
in which they are suspended & are thus difficult to see by bright
field techniques,due to their lack of contrast.

51. • Direct ground microscopy
overcomes these problems by
preventing direct light from
entering the front of objective
& the only light gathered is
that reflected or diffracted by
structures within specimen.this
causes specimen to appear as a
bright image on a dark
background,the contrast being
reversed & increased.

52. • In dark ground microscopes,the objective must have a lower
numerical aperture than condenser(in bright field illumination
,optimum efficiency is obtained when NA of both objective &
condenser are matched).To obtain this condition it is sometimes
necessary to use objectives with built in iris diaphragm or by
inserting a funnel stop into objective.
• Perfect centering of condenser is essential & with oil immersion
systems it is necessary to put oil between condenser & object slide
in addition to oil between slide & objective.
• As only light diffracted by specimen will enter the objective ,a
high intensity light source is required.

53. • Most bright field microscopes can be converted for dark ground
work by using simple patch stops made of black paper placed on
top of condenser lens or suspended in the filter
holder.alternatively,the patch stops can be constructed from
different colored filters (rheinberg illumination)using a dark color
for center disc & a contrasting lighter code for periphery.this
system reduces glare of conventional dark ground
• Dark ground illumination is useful for spirochetes,flagellates,cell
suspensions,parasites.
• Many small structures are more easily visualised by dark ground
techniques due to increased contrast ,although resolution may be
inferior to bright field microscopy.

54. PHASE CONTRAST MICROSCOPE
• Professor Zernicke was awarded the nobel prize for his work on
phase contrast.
• Is an optical microscopy illumination technique in which small
phase shifts in the light passing through a transparent specimen
are converted into amptitude or contrast changes in the image

55. • Two rays of light from the same source having same frequency –
coherent & when recombined will double in amplitude or
brightness – if in phase with each other-CONSTRUCTIVE
INTERFERENCE.
• If out of phase with each other-DESTRUCTIVE INTERFERENCE
will occur.

56. Represents wave form of light ray
Rays are identical but one is
1 λ out of phase
4
They interfere with no increase in
amplitude

57. One ray 1 λ out of phase & cancel out each other
2
No light, maximum interference,maximum
contrast
One ray brighter than other(increased
amplitude,difference in amplitude can be
seen,maintaing maximum interference-occurs in
phase contrast microscope.

58. • To achieve phase contrast
microscope requires modified
objectives & condenser & relies on
specimen retarding light by
1 & 1 λ
8 4
• Microscope condenser usually carries
a series of annular diaphragms
made of opaque glass,with a clear
narrow ring ,to produce a
controlled hollow cone of light.
• Each objective requires a different
size of annulus ,an image of which is
formed by condenser in back focal
plane of objective as a bright ring of
light.
• Objective is modified by a phase
plate consisting of a clear glass disc
with circular trough etched in it to
half the depth of disc.

59. • The light passing through trough
has a phase difference of 1λ
4
compared to rest of the plate.
• The trough also contains neutral
density light absorbing material to
reduce brightness of direct
rays,which would otherwise obscure
the contrast obtained.

60. • It is essential that image of bright
annular ring from condenser is centered
& superimposed on the dull trough of
objective phase plate(obtained by using a
focusing telescope in place of eyepiece or
a bertrand lens in body tube.
• When hollow cone of direct light from
annulus enters specimen some will pass
through unaltered while some rays will
be diffracted/retarded by approx 1λ
4

61. • The direct light will mostly pass
through trough in phase plate while the
diffracted rays pass through the thicker
clear glass & further retarded.
• The total retardation of the diffracted
rays is now 1λ
2
and interference will occur when they
are recombined with direct light.
• Thus an image of contrast is achieved
revealing small details within unstaind
cells.
• Quick & efficient way of examining
unstained paraffin,resin & frozen
sections & studying living cells.

62. INTERFERENCE MICROSCOPY
• In Phase contrast microscope, the specimen retards some light
rays with respect to those with pass through the surrounding
medium , the resulting interference will provide a contrast
but with an artefact called the “Phase Halo”.
The retarded rays in interference microscopy are entirely
separated from direct or reference rays; so provide:-
1. Improved image contrast
2. Color graduation.
3. Better Section thickness

63. with monochromatic light the bands are
alternately dark & light & of a single color.the
Same effect can be shown if separate beam
of coherent light are reunited.this
phenomenon is known as INTERFERENCE
Two slits closely side by side form two fans of
rays which cross & if coherent will interfere.if
each ray regarded as wave-phase conditions of
increased amplitude & extinction occur at
points where waves cross & interfere.the result
is series of parallel bands alternately bright &
dark across field of view.with white light
,bands of spectral colors seen(wavelengths
making white color refracted at different
angles).

64. • Used double beam system where the
separation is produced by birefringent
material & is close enough to require
only one objective.
• If two paths are equal & in same phase
,interference bands can be seen running
straight & parallel across the field.
• If into one beam path an object is
introduced –causes shift in interference
bands.
• Displacement of bands is measured with
a micrometer eyepiece & this
information coupled with RI or object
thickness –measurements determined.

65. POLARIZED LIGHT MICROSCOPY
• Polarised light microscopy – diagnostic.
• Natural light vibrates in many planes,POLARISED LIGHT VIBRATES
IN ONLY ONE PLANE.
• Numerous crystals,fibrous structures,pigments,lipids,proteins,bone &
amyloid deposits exhibit BIREFRINGENCE.
• Crystals capable of producing polarised light are called
BIREFRINGENT
• Can be produced for microscopy purpose by passing natural light
through a polariser..

66. • When a ray of light will pass through the unevenly dense
crystal(LIKE CALCITE)will retard the two rays to a differing
degree and since refraction is partly dependent on density, the
two rays will be refracted or bent to differing degrees. This is
known as Double Refraction Or Birefringence.

67. • A ray of light entering such a
crystal will be converted into
2 rays which will emerge at
different points, and the
emergent light ray will be
polarized light. i.e., the all the
vibrations in one ray will be
in one single direction and
other ray in other single
direction ; but these
directions will be at right
angles to each other.

68. TYPES OF BIREFRINGENCE
1. Intrinsic or Crystalline Birefringence
2. Form birefringence
3. Strain birefringence
4. Positive birefringence
5. Negative birefringence

69. Intrinsic / crystalline birefringence
Type of anisotropy which occurs due to an asymmetrical
arrangement of chemical bonds, ions or molecules
• Biological objects exhibiting this birefringence are
• Collagen
• Muscle fiber
• Chromosomes.

70. Form Birefringence
Found in mixed bodies, wherein asymmetrical particles of one
refractive index are dispersed in a specially oriented manner in a
medium having a different refractive index. At least one
dimension of particle must be small in relation to wavelength of
light employed.these dispersed particles may be filaments ,sheets
& may be dispersed in liquid,gas or solid.(examining objects
mounted in a variety of mountants with differing RI for e.g
water,glycerol).

71. Strain Birefringence
When a dielectric substance is subjected to mechanical stress, the
bonds within the substance can be distorted and give rise to a
pattern which will result in birefringence.
Eg. Elastic tissue fibers under stress show birefringence.

72. SIGN OF BIREFRINGENCE
• If the slow ray (higher RI) is parallel to length of crystal or fibre
BIREFRINGENCE IS POSITIVE.
• If slow ray is perpendicular to long axis of structure
BIREFRINGENCE IS NEGATIVE.
• Diagnostically useful & determined by use of
compensator(birefringent plate of known retardation) either
above the specimen or below the polariser at 45 degree to
direction of polarised light.
• Rotate compensator or specimen until slow direction of
compensator(indicated by arrow) is parallel to long axis of crystal
or fiber.

73. o Quartz & collagen exhibit positive birefringence.
o Calcite,urates & chromosomes exhibit negative birefringence.
o Simple compensators can be made from mica or layers of
sellotape.
The field is now red.

74. APPLICATION TO MICROSCOPY
Polarizing microscopes uses two polarizer
Polarizer
• Placed beneath the substage condenser
• Held in rotatable graduated mount
• Can be removed from light path when not required

75. Analyzer
• Placed between objective and eye-piece
• When we look through a single polarizer, there is loss of intensity
which can be because of
Colour of filter
Splitting of rays
Absorption of rays.

77. When we look through 2 polarizer, there are 3 conditions:-
– When polarizer and analyzer are parallel
Rays vibrating in the parallel plane are able to pass.
- When polarizer and analyzer are crossed, rays able to pass
the polarizer are blocked by analyzer.
The condition when no light reaches to the observer is
known as Extinction.

78. - When a birefringent substance is rotated between crossed polarizer, it
is visible when it is in the diagonal position. (i.e., when it is halfway (45º)
between the vibration planes of polarizer).
Extinction occur when one of its planes of vibration is
parallel to either polarizer.
(Both these conditions occurs 4 times in 360º revolution).

79. substances can produce plane polarised light by differential absorption & give rise to
phenomenon -DICHORISM

80. FLUORESCENCE MICROSCOPY
• Fluorescence is the property of some substances which when
illuminated by light of a certain wavelength will re-emit the light
at a longer wavelength.
• In fluorescence microscopy the exciting radiation is usually in
ultra violet wavelength(360 nm) or blue region (400 nm).
• A substance which possesses a FLUOROPHORE will fluoresce
naturally known as PRIMARY FLUORESCENCE or
AUTOFLUORESCENCE.

81. • Ultraviolet excitation is required for optimum results with
substances such as vitamin A,porphyrins and chlorophyll.
• dyes.,chemicals & antibiotics added to tissues produce
SECONDARY FLUORESCENCE of structures & are called
FLUOROCHROMES(most common use of fluorescence microscopy
& majority of fluorochromes require only blue light excitation).
• INDUCED FLUORESCENCE is term applied to substances such as
catecholamines which after treatment with formaldehyde vapor
are converted to fluorescent quinoline compounds.

82. FLUORESCENCE
TRANSMITTED LIGHT FLUORESCENCE INCIDENT LIGHT FLUORESCENCE

83. TRANSMITTED LIGHT FLUORESCENCE
• Most commonly used are HIGH PRESSURE GAS LAMPS such as
MERCURY VAPOR & XENON GAS LAMPS.
• For wavelength excitation in blue & green range halogen filament
lamps are useful.
• Mercury vapour burners operate on alternating current.
• Xenon burners operate on direct current.which require rectifiers.

84. • Mercury vapor & xenon burner lamps differ in emission curves
.mercury lamps at some wavelengths reach very high amplitudes
whereas at other parts of wavelengths range the emission is low-
curve-spiky profile.peaks coincide with excitation wavelengths of
more widely used fluorochromes.
• Xenon has smoother continuous curve

85. FILTERS
• Filter out specific excitation
wavelength to avoid
confusion between
important & unimportant
fluorescence.
• ‘dyed in the glass’ filters –
designated as UG 1 & BG
12- these are broad band
filters & transmit wide
range of
wavelengths(depending on
composition & thickness of
filter).

86. • narrow band filters(for materials
excited at more than one
wavelengths)-vacuum coated
layers of metals on glass
support.They have a mirror like
surface & must be inserted in
beam with reflective face towards
light source.
• Barrier/suppression filters are
placed before eyepiece to prevent
short wavelength light from
damaging retina of eye.K.470
filter will block all wavelengths
below 470 nm.

87. CONDENSERS FOR FLUORESCENT MICROSCOPY
• Bright field condensers are able to illuminate the object using
all available energy but also direct rays beyond object into
objective-potential hazard to eyes of observer & can set up
disturbing autofluorescence in cement & component layers of
objective.
• Most systems employ a dark ground condenser which does not
allow direct light in objective & more certain to give a dark
contrasting background to fluorescence.

88. OBJECTIVES IN FLUORESCENT MICROSCOPE
• With bright field illumination(autofluorescence hazardous)-
Simpler achromat objectives are practical.
• With dark ground illumination-range of objectives widened-
elaborate lenses with higher apertures & better gathering power
are possible.

89. INCIDENT LIGHT FLUORESCENCE
• The excitation beam after passing
the selection filters is diverted
through objective on preparation
where fluorescence is stimulated.this
fluorescence travels back to observer
by normal route.
• Dichoric mirrors have been
produced to divide & divert the
beam.These mirrors have the
property of being able to transmit
light of some wavelength & reflect
others.
a. diagram of incident
fluorescence microscope
layout
b. b. effect of dichoric mirror

90. • By selection of appropriate mirror the wavelength desired is
reflected to object the remainder passes through to be lost.
• Visible fluorescent light collected by objective in normal way
can pass to eyepiece & any excitation rays bouncing back
from slide & coverglass are reflected back along original path
to source.thus prevented from reaching the observer.

91. • The objective acts as condenser-illumination & objective
numerical aperture are same.-efficient condition.
• Fluorescence is stimulated on observers side of
preparation & therefore more brilliant –not masked by
covering material or section thickness.
• Use of dichoric mirrors –much brighter images.,since
upto 90% of exciting energy can reach the preparation
& 90% of resultant visible light can be presented to eye.

92. CLEANING THE MICROSCOPE
• Materials used-
o Special lens tissue paper.
o A piece of chamois leather.
o Soft camel hair brush.
o A desiccator 15-20 cm in dia containing not less than 250 gm
of dry blue silica gel(indicates humidity by turning pink).

93. CLEANING OF OPTICAL SURFACES
• Fine paint brush or camel hair brush.
• Oil residues on lens should be removed with special tissue
paper,absorbent paper or medical grade cotton wool.
Solution-80 % petroleum ether
-20 % 2-propanol
 Do not use 95% ethanol,xylene or toluene for cleaning lenses since
these substances dissolve the cement.Can be used for cleaning
mirrors.

94. Thank you