microscopy in pathology

1. DIFFERENT VARIANTS OF
MICROSCOPE
Dr Uttam Kumar Das
PGT-1 Dept Of Pathology
BSMC Bankura

2. BASICS
 Visible Light:
Electromagnetic spectrum detected by human
eye- 400nm(deep violet) to 800nm(Far red).
Human eye doesn’t see much beyond
700nm(deep red).
 White light:
Mixture of light containing some % of
wavelength from all visible portion.

3. BASICS
 Color Temperature:
Measure of mixture of light given off by light
source.
Higher the color temp closer is to natural
daylight.
Natural daylight color temperature~5200kelvin
Incandescent light from Tungsten Bulb~3200k
Higher color temp- more blue/white to eye.
Lower color temp- more red to yellow to eye &
regarded as being ‘warmer’ in color.

4. BASICS
 The Amplitude (i.e. brightness):
Decreases as light gets further from source
because of absorption in media it passes.
 Energy Level:
Energy content of light, expressed as electron
volt per photon (the particle representation of
light).
Visible light-1electron volt/photon.
Soft x-ray portion-50-100ev/photon
Energy increases moving towards violet &
ultraviolet range of spectrum.

5. BASICS
 Spherical aberration:
Light rays hitting periphery will be more
refracted than the rays hitting centre of lens.
 Chromatic Aberrations:
White light of passing through simple lens, each
wavelength will be refracted to different extent.
Blue brought to a shorter focus than red, results
in a un-sharp image with color fringes.
Corrected with ‘Achromat’ & ‘Apochromat’ lens.

6. BASICS
 Magnification:
For variable tube length=From 160-250mm
Optical tube length X Magnification of eye piece
Focal length of the objective
 Resolving Power:
Capacity of the optical system to produce
separate images of objects very close to each
other.
Resolving power of std LM=200nm

7. BASICS
 Illumination:
Critical illumination-when the light source is
focused by condensor in the same plane as the
object, when the object is in focus.
The illuminating light consists of rays or waves
that are nearly parallel to one another.
 This quality of light is called partially coherent
light. The ultimate resolving power of the
microscope depends on the use of partially
coherent illumination.
 The condenser in Köhler illumination is used to
create and focus this light onto the specimen

8. BASICS
 Real Image:
The real image forms on the side of the lens
opposite the object.
A slide projector produces a real image on a
projection screen.
The objective lens produces a real image in
the intermediate image plane.

9. BASICS
 Virtual Image:
A virtual image cannot be seen on a screen. A
second lens is required to see a virtual image and
this would cause the image to be inverted
relative to the specimen.
To a human, the virtual image appears to be on
the same side of the lens as the object.
The image we see in a compound microscope is a
virtual image.
In upright microscopes the virtual image is
inverted. In inverted microscopes the image is
made to be upright by optics in the binocular.

10. BASICS
 Back Focal Plane:
The side of a lens where an image is formed is called
the image side or back side of the lens. The plane at
one focal length on this side is the back focal plane.
The back focal plane of the objective lens is on
the inside of the microscope tube.
 Coma:
Resulting in the images of structures out from the
center being smeared outwards.
 Astigmatism:
Resulting in light in the X plane being focused
differently to light in the Y plane.

12. •NUMERICAL APARTURE:
• Is a dimensionless number that characterizes
the range of angles over which the system can
accept or emit light.
•It is the acceptance cone of an objective (and
hence its light-gathering ability and resolution).
The numerical aperture
with respect to a
point P depends on the
half-angle θ of the
maximum cone of light
that can enter or exit the
lens.
where n is the index of refraction of the medium
in which the lens is working, and θ is the half-
angle of the maximum cone of light that can
enter or exit the lens
BASICS

13. BASICS
 Compound Lenses:
There are four basic lens shapes: double
convex, double concave, plano convex and
plano concave.
In the 1830’s, the solution to chromatic
aberrations was found by cementing two lenses
made of different glasses (one Double convex and
one Plano-concave) together into an Achromatic
Doublet to make a compound lens.
Today, compound lens consists of many lens
elements. These elements are necessary for
magnification and correction of various lens
aberrations.

14. REFRACTIVE INDEX
•Refractive index is the
light-bending ability of a
medium.
• The light may bend in air
so much that it misses the
small high-magnification
lens.
• Immersion oil is used to
keep light from bending.
RI of Air- 1.0
RI of Water -1.3
RI of Glycerol- 1.47
RI of Glass(avg)- 1.5
RI of Oil- 1.52

15. UNITS OF MEASUREMENT
 1 µm micrometer = 1/1000 mm
 1 nm nanometer = 1/1000 µm
 Parfocal distance: Distance between objective
shoulder and specimen(45 mm for most
microscopes).
 Working Distance Focal Length
Scanning- 17-20mm 40mm
Low power- 4-8mm 16mm
High power- 0.5-0.7mm 4mm
Oil immersion- 0.1mm 1.8-2.0mm

20. INFINITY CORRECTED MICROSCOPES
 A majority of modern research microscopes are
equipped with infinity-corrected objectives that
no longer project the intermediate image directly
into the intermediate image plane. Light
emerging from these objectives is instead focused
to infinity, and a second lens, known as a tube
lens, forms the image at its focal plane.

21. INFINITY CORRECTED MICROSCOPES
 Wavetrains of light leaving the infinity-focused
objective are collimated, allowing beam-splitters,
polarizers, Wollaston prisms, etc., to be
introduced into the space between the objective
and tube lens.

22. INFINITY CORRECTED MICROSCOPES
 In a microscope with infinity-corrected optics,
magnification of the intermediate image is
determined by the ratio of the focal lengths of the
tube lens and objective lens.
 Because the focal length of the tube lens varies
between 160 and 250 millimeters (depending
upon the manufacturer and model), the focal
length of the objective can no longer be assumed
to be 160 millimeters divided by its
magnification.
 Thus, an objective having a focal length of 8
millimeters in an infinity-correct microscope with
a tube lens focal length of 200 millimeters would
have a lateral magnification of 25x (200/8).

23. MODERN TECHNOLOGY IMPROVING MICROSCOPY
 The invention of the microscope allowed scientists and
scholars to study the microscopic creatures in the world
around them.
 When learning about the history of the microscope it is
important to understand that until these microscopic
creatures were discovered, the causes of illness and
disease were theorized but still a mystery.
 The microscope allowed human beings to step out of the
world controlled by things unseen and into a world where
the agents that caused disease were visible, named and,
over time, prevented.

24. MICROSCOPES CAN BE SEPARATED INTO SEVERAL
DIFFERENT CLASSES
 One grouping is based
on what interacts with
the sample to generate
the image:
-Light or photons(optical
microscopes)
-Electrons (electron
microscopes)
-Probe (scanning probe
microscopes).
 Whether they analyze
the sample via:
-Scanning point (confocal
optical microscopes,
scanning electron
microscopes and scanning
probe microscopes)
-Analyze the sample all at
once (wide field optical
microscope and
transmission electron
microscopes).

25. BRIGHT-FIELD MICROSCOPY
 The useful magnification of Light microscope is
limited by its resolving power.
 The resolving power in limited by wavelength of
illuminating beam.
 Resolution is determine by certain physical
parameters like wave length of light and light
generating power of the objective & condenser lens.
 Higher N.A Better light generation Better Resolution
Shorter the Wavelength Better Resolution.
 The ordinary microscope is called as a bright field
microscope. It forms dark image against bright
background.

26. Dark objects are visible
against a bright
background.
Light reflected off the
specimen does not
enter the objective lens.

27. ADVANTAGES
 Bright field compound microscopes are commonly
used to view live and immobile specimens such as
bacteria, cells, and tissues.
 For transparent or colorless specimens, however,
it is important that they be stained first so that
they can be properly viewed under this type of a
microscope.
 Staining is achieved with the use of a chemical
dye. By applying it, the specimen would be able
to adapt the color of the dye. Therefore, the light
won’t simply pass through the body of the
specimen showing nothing on the microscope’s
view field

28. DARK FIELD MICROSCOPY

30. PRINCIPLE
 When observing unstained/living cells, such specimens & their
components have refractive indices close to that of the medium
in which they are suspended & are thus difficult to see by bright
field techniques, due to their lack of contrast.
 Dark field microscopy overcomes these problems by
preventing direct light from entering the front of the
objective & the only light gathered is the reflected or
diffracted by structures within the specimen.
This causes the specimen to appear as a bright image on
a dark background, the contrast being reversed &
increased.
 Dark field permits the detection of particles smaller than the
optical resolution that would be obtained in bright field, due to
high contrast of scattered light.
 In the microscope, oblique light is created by using a
modified or special condensor that form a hollow cone of
direct light which will pass through the specimen but
outside the objective.

31. DARK FIELD MICROSCOPY
 Light enters the microscope for illumination of
the sample.
 A specially sized disc, the patch stop blocks
some light from the light source, leaving an
outer ring of illumination.
A wide phase annulus can also be reasonably
substituted at low magnification.
 The condenser lens focuses the light towards the
sample.

32. DARK FIELD MICROSCOPY
 The light enters the sample. Most is directly
transmitted, while some is scattered from the
sample.
 The scattered light enters the objective lens,
while the directly transmitted light simply
misses the lens and is not collected due to
a direct illumination block.
 Only the scattered light goes on to produce the
image, while the directly transmitted light is
omitted.

34. DARK FIELD MICROSCOPY
 Dark field microscopy is a very simple yet
effective technique and well suited for uses
involving live and unstained biological samples:
-Spirochetes
-Flagellates
-Cell Suspension
-Flow Cell Techniques
-Parasites
-Autoradiographic grain counting
-Once used in Fluorescence Microscopy

35. DARK FIELD MICROSCOPY
 In Dark Field Illumination the objective must
have a lower Numerical Aperture than the
condensor.
 In Bright Field Illumination optimum
efficiency is obtained when the Numerical
Aperture of both the Objective and the Condensor
are matched.
 Dark Field Condensor may be:
-Dry, low power objective
-Oil immersion, high power objective

36. ADVANTAGES
 The advantage of darkfield microscopy also becomes
its disadvantage: not only the specimen, but dust and
other particles scatter the light and are easily
observed For example, not only the cheek cells but the
bacteria in saliva are evident. The dark field
microscopes divert illumination and light rays thus,
making the details of the specimen appear luminous.
 Dark field light microscopes provide good results,
especially through the examination of live blood
samples. It can yield high magnifications of living
bacteria and low magnifications of the tissues and
cells of certain organisms. Certain bacteria and fungi
can be studied with the use of dark field microscopes.

37. THANKYOU

38. PHASE CONTRAST MICROSCOPY

39. DEFINITION
 Phase contrast microscopy is an optical
illumination technique in which small phase
shifts in the light passing through a transparent
specimen are converted into amplitude or
contrast changes in the image. The technique
was invented by Frits Zernite in the 1930s for
which he received the Nobel prize in physics in
1953 .

40. Accentuates
diffraction of
the light that
passes through
a specimen.
Direct and
reflected light
rays are
combined at the
eye. Increasing
contrast
PHASE CONTRAST MICROSCOPY

41. APPLICATION
 Applications for phase contrast microscopy
equipment range from the study of living
biological specimens, medical applications, study
of live blood cells, and other biological and science
applications.
 Most commonly used to provide contrast of
transparent specimens such as living cells or
small organisms.
 Useful in observing cells cultured in vitro during
mitosis.

42. PHASE CONTRAST MICROSCOPY
 Unstained/living biological specimens have little
contrast with their surrounding medium.
To see them clearly involves:
A-Closing down the iris diaphragm of condensor
which decreases the Numerical Aperture producing
diffraction effects & destroy the Resolving Power of
the Objective.
B-Or when using dark field illumination, which
enhances contrast by reversal, but often fails to reveal
internal details.
 The Phase Contrast microscopy overcomes these
problems by controlled illumination using the full
aperture & thus improving the Resolution.
The high the refractive index of the structure, the
darker it will appear against the light background,
i.e. with more contrast.

43. PHASE CONTRAST MICROSCOPY
 If a diffraction grating is examined under the
microscope, diffraction spectra are formed in the
Back Focal Plane of the objective due to
interference between the direct & diffracted rays
of light.
 The grating consists of alternate strips of
material with slightly different refractive indices,
through which light acquire small phase
difference & these form the images.

45. PHASE CONTRAST MICROSCOPY
 By converting the phase differences, between
light passing through a specimen and that
passing through the surrounding medium, into
amplitude (brightness) differences, phase
contrast microscopy provides a difference in
brightness between the object and the
background, which the eye can then see.

46. PHASE CONTRAST MICROSCOPY
 Phase contrast microscopy uses an annular stop in
the condenser and a phase plate within the objective
lens, which is aligned with the annular stop.
In this configuration, the light path can be split and
each of the separated beams will pass through the
same transparent medium at the specimen stage.
The light passes through the annular stop and forms
a cone of light, which comes to its vertex at the focal
point of the specimen.
Any background light, which is un-deviated by the
specimen, will go through the phase ring in the phase
plate and is advanced by a quarter of a
wavelength. Deviated light passing through the
specimen is retarded by a quarter of a wavelength
and passes through the phase plate without going
through the ring.

47. PHASE CONTRAST MICROSCOPY
 When the beams are recombined further along
the light path, the differences in the phase of the
deviated and un-deviated light beams become
additive and subtractive.
 The resultant wave is the sum of the two waves
which have their crests and troughs opposite
each other. The difference in amplitude can be
seen as a change in brightness, since brightness
is proportional to the square of the amplitude.
The net result is that features of the object are
either lighter or darker than the surrounding
field.

50. Plane Light
Polarized Light
High Relief

52. HOW IS LIGHT POLARIZED

53. PRINCIPLE
 Light is a series of pulse of energy radiating away from
a source & shown diagrammatically as sine curve,
with wavelength & amplitude.
 Light can also be described as electromagnetic
vibration, which travels outwards from source of its
propagation, much in the same way as vibration along
a rope.
 Natural light vibrates in many directions but polarized
light in only one direction.

54. POLARIZED LIGHT MICROSCOPY
 Polarization can be achieved with the use of
substance/crystals which allow vibration only in one plane
& called birefringent.
 Light entering a birefringent crystal such as calcite is split
into two light paths, each determined by a different
refractive index & each vibrating in one direction only, but
at right angle to each other.
 The higher the RI, the greater the retardation of the ray, so
that each ray leaves the crystal at a different velocity.
 The high RI ray is called slow and low RI ray is called
fast.
 There is also a phase difference between the rays, so that if
they are recombined interference occurs & various spectral
colors are seen.

55. POLARIZED LIGHT MICROSCOPY
 Two discs made up of prism are placed in a path
of light, one below the objective known as
Polarizer & another placed in the body tube
which is known as Analyzer.
 Polarizer sieves out ordinary light rays vibrating
in all directions allowing light waves of one
orientation to pass through.
 The lower disc (polarizer) is rotated to make the
light plane polarized. During rotation, when
analyzer comes perpendicular to polarizer, all
light rays are cancelled or extinguished.
 Birefringent objects rotate the light rays &
therefore appear bright in a dark background.

56.  Analyzer (upper polarizer) -- a
polarizing prism located above the
microscope stage, between the
objective lens and the eyepiece. This
restricts the transmission of light
vibrating perpendicular to the
polarizer. The analyzer can be
slipped in or out of the light path or
rotated for partially crossed polarized
light. Light passing through the
polarizer will not pass through the
analyzer unless the vibration
direction of the light is changed
between the two prisms. Anisotropic
minerals can perform this deed.
 Polarizer (lower polarizer) -- a
polarizing prism located beneath the
microscope stage (between the light
source and the object of study). This
restricts transmission of light to that
vibrating in only one (N-S) direction.
Some microscopes have a different
orientation direction. In effect, it
plane polarizes the incident light
beam.

57. POLARIZED LIGHT MICROSCOPY
 Numerous crystals, fibrous structures (both natural &
artificial), pigment, lipid, protein, bone & amyloid
deposits exhibit birefringence.
 Originally, polarizer's, made from calcite & known as
Nicol Prism, were cemented together with Canada
balsam in such away that the slow rays was deflected
away from the optical path & into the mount of the prism,
leaving only the polarized fast rays to pass through (optic
axis).
 Some substances & crystals can produce plane polarized
light by differential absorption & give rise to phenomenon
of Dichorism. These absorb the slow rays & are pleochoric
(absorbing all colors equally) and are the most useful in
microscopy.
Such crystals suspended in thin plastic films & oriented in
one direction have replaced bulky Nicol Prism.

58. POLARIZED LIGHT MICROSCOPY
 Two phenomenon detected in polarized light-
Birefringence & Dichroism.
 When a birefringent substance rotated between two
crossed polarizers, the image appears & disappears
four times each in 360 deg rotation (at 45̊ each).
 Dichroism- Only the polarizer is used &, if no
rotatory stage is available the polarizer itself can be
rotated. Changes in intensity & color is seen during
rotation at 90 deg. This is due to differential
absorption of light, depending upon the vibration
direction of the two rays in a birefringent substance.
 Weak birefringence in biological specimen is
increased by addition of dyes or impregnating metals,
in a orderly linear alignment e.g. amyloid fibrils.

59. DIFFERENTIAL INTERFERENCE CONTRAST

60. DIFFERENTIAL INTERFERENCE
CONTRAST
 In this complex form of polarised light microscopy two
slightly separate, plane polarised beams of light are
used to create a 3D-like image with shades of grey.
 Wollaston prisms situated in the condenser and
above the objective produce the effect, and additional
elements add color to the image.
 Care must be taken to interpreting DIC images as the
apparent hills and valleys in the specimen can be
misleading. The height of a "hill" (e.g. the nucleus) is
a product of both the actual thickness of the feature
(i.e. ray path length) and its refractive index.
 Variations of the DIC system are named after their
originators, Nomarski and de Senarmont. Options
can be selected to maximize either resolution or
contrast.

61. Accentuates
diffraction of
the light that
passes through
a specimen;
uses two beams
of light. Adding
color
Differential Interference Contrast Microscopy

67. ELECTRON MICROSCOPY

68. INTRODUCTION
 Electron microscope is a type of microscope that
uses a particle beam of electrons to illuminate a
specimen & create a highly-magnified image. Co-
invented by Germans, Max Knoll and Ernst
Ruska in 1931

69. ELECTRON GUN
 2 types of guns are used in electron microscopy-
Thermionic Emission Gun & Field Emission Gun.
 Thermionic: Electrons emitted from heated filament (
tungsten, Lanthanum Hexaboride). Most common, cheap &
ultra high vacuum not required.
 Field Emission: Strong electron field used to extract
electrons from filament. High vacuum needed.

70. ELECTROMAGNETIC LENS
 An electromagnet designed to produce a suitably
shaped magnetic field for focusing &
deflection of electrons in electron optical
instruments.
 A strong magnetic field is generated by passing a
current through a set of windings.
 This field acts as a convex lens in case of electron
microscope.

71. CONDENSER LENS
 The first lens(controlled by "spot size knob")
largely determines the "spot size"; the general
size range of the final spot that strikes the
sample.
 Second condenser lens: The second
lens(controlled by the "intensity/ brightness
knob" changes the size of the spot on the sample;
changing it from a wide dispersed spot to a
pinpoint beam.

72. OTHER PARTS
 The other parts include:
-Condenser aperture,
-Objective lens,
-Objective aperture,
-Selected area aperture(to examine diffraction
patterns),
-Intermediate lens(magnifies initial image formed
by objective lens) &
-Projector lens.

73. TYPES OF ELECTRON MICROSCOPES
 There are 2 types of electron microscopes:
 Transmission Electron Microscope: Process
is carried out under vacuum to avoid friction. The
"Virtual Source" at the top represents
the electron gun, producing a stream of
monochromatic electrons. The usual potential is
around 10000 – 15000V.
 SCANNING ELECTRON MICROSCOPE

74. TRASMISSION ELECTRON
MICROSCOPY

75. •Ultrathin sections of specimens.
•Light passes through specimen, then an
electromagnetic lens, to a screen or film.
•Specimens may be stained with heavy metal salts.
Transmission Electron Microscopy (TEM)

76. TRANSMISSON ELECTRON MICROSCOPE
 This transmitted portion is focused by the objective
lens into an image.
 The Objective & Selected Area
metal apertures restrict the beam.
 The image is passed down the column through the
intermediate and projector lenses, being enlarged all
the way.
 The image strikes the phosphor image screen & light
is generated, allowing the user to see the image.
 The darker areas represent areas that fewer
electrons were transmitted (thicker or denser). The
lighter areas represent areas that more electrons
were transmitted (thinner or less dense)

77. TRANSMISSION ELECTRON MICROSCOPE
 Transmission electron microscopy (TEM) involves a high voltage
electron beam emitted by a cathode and formed by magnetic
lenses. The electron beam that has been partially transmitted
through the very thin (and so semitransparent for electrons)
specimen carries information about the structure of the
specimen.
 The spatial variation in this information (the "image") is then
magnified by a series of magnetic lenses until it is recorded by
hitting a fluorescent screen, photographic plate, or light
sensitive sensor such as a CCD (charge-coupled device) camera.
The image detected by the CCD may be displayed in real time on
a monitor or computer.
 Transmission electron microscopes produce two-dimensional,
black and white images.
 Resolution of the TEM is also limited by spherical and chromatic
aberration, but a new generation of aberration correctors has
been able to overcome or limit these aberrations.

80. SCANNING ELECTRON
MICROSCOPE

81. •An electron gun produces a beam of electrons that scans the
surface of a whole specimen.
•Secondary electrons emitted from the specimen produce the
image.
Scanning Electron Microscopy (SEM)

82. SCANNING ELECTRON MICROSCOPE
 Unlike the TEM, where the electrons in the primary beam
are transmitted through the sample, the Scanning Electron
Microscope (SEM) produces images by detecting secondary
electrons which are emitted from the surface due to
excitation by the primary electron beam.
 In the SEM, the electron beam is scanned across the
surface of the sample in a raster pattern, with detectors
building up an image by mapping the detected signals with
beam position.

85. SCANNING ELECTRON MICROSCOPE
 The 1st scanning electron microscope (SEM)
debuted in 1938 by Von Ardenne with the first
commercial instruments out around 1965.
 In this case electrons are not used to directly
image the specimen, but to excite it in such a way
that it gives out secondary electrons which are
collected by detectors & used to form the image.
 The first scanning electron microscope (SEM)
debuted in 1938 ( Von Ardenne) with the first
commercial instruments around 1965.

86. SPECIMEN
 For electron microscopy, tissue is fixed in 4%
glutaraldehyde at 4°C for 4 hours.
 Fixation - In chemical fixation for electron
microscopy, glutaraldehyde is often used to crosslink
protein molecules and osmium tetroxide to preserve
lipids.
 Dehydration - Organic solvents such as ethanol or
acetone for SEM specimens or infiltration with resin
and subsequent embedding for TEM specimens.
 Embedding - Resin (for electron microscopy) such as
araldite or LR White, which can then be polymerised
into a hardened block for subsequent sectioning.

87. SPECIMEN
 Sectioning - Typically around 90nm cut on an
ultramicrotome with a glass or diamond knife. Glass
knives can easily be made in the laboratory and are
much cheaper than diamond, but they blunt very
quickly and therefore need replacing frequently.
 Staining - uses heavy metals such as lead and
uranium to scatter imaging electrons and thus give
contrast between different structures, since many
(especially biological) materials are nearly
"transparent" to the electron beam.
 By staining the samples with heavy metals, electron
density is added to it which results in there being
more interactions between the electrons in the
primary beam and those of the sample, which in turn
provides us with contrast in the resultant image.

88. ARTIFACTS
 It must be emphasized from the outset that every
electron micrograph is, in a sense, an artifact.
 Artifacts present themselves in many ways:
-There could be loss of continuity in the
membranes,
-Distortion or disorganization of organelles,
-Empty spaces in the cytoplasm of cells or
sharp bends or curves in filamentous structures
that are usually straight, such as microtubules
and so on.
 With experience, microscopists learn to recognize
the difference between an artifact of preparation
and true structure.

89. DISADVANTAGES OF ELECTRON MICROSCOPY
 Electron microscopes are very expensive to buy and maintain.
 They are dynamic rather than static in their operation: requiring
extremely stable high voltage supplies, extremely stable currents to
each electromagnetic coil/lens, continuously-pumped high/ultra-high
vacuum systems and a cooling water supply circulation through the
lenses and pumps.
 As they are very sensitive to vibration and external magnetic
fields, microscopes aimed at achieving high resolutions must be
housed in buildings with special services.
 A significant amount of training is required in order to operate
an electron microscope successfully and electron microscopy is
considered a specialised skill.
 The samples have to be viewed in a vacuum, as the molecules that
make up air would scatter the electrons. This means that the samples
need to be specially prepared by sometimes lengthy and difficult
techniques to withstand the environment inside an electron
microscope.
 Recent advances have allowed some hydrated samples to be imaged
using an environmental scanning electron microscope, but the
applications for this type of imaging are still limited.

90. CONFOCAL MICROSCOPY

91. CONFOCAL MICROSCOPY

92. PRINCIPLE
 Confocal microscopy is an optical imaging
technique used to increase optical
resolution and contrast of a micrograph by using
point illumination and a spatial pinhole to
eliminate out-of-focus light in specimens that are
thicker than the focal plane.
 It enables the reconstruction of three-
dimensional structures from the obtained images.

93. •Uses fluorochromes
and a laser light.
•The laser illuminates
each plane in a
specimen to produce a
3-D image.
Confocal Microscopy

94. INTRODUCTION
 Although conventional light and fluorescence microscopy
allow the examination of both living and fixed specimens,
certain problems exist with these techniques.
-One of the main problems is out-of-focus blur degrading
the image by obscuring important structures of interest,
particularly in thick specimens.
 In conventional microscopy, not only is the plane of focus
illuminated, but much of the specimen above and below
this point is also illuminated resulting in out-of-focus blur
from these areas.
 This out-of-focus light leads to a reduction in image
contrast and a decrease in resolution.

95. APPLICATIONS IN CONFOCAL MICROSCOPY
 The broad range of applications available to laser
scanning confocal microscopy includes a wide
variety of studies in neuroanatomy and
neurophysiology, as well as morphological studies
of a wide spectrum of cells and tissues.
 In addition, the growing use of new fluorescent
proteins is rapidly expanding the number of
original research reports coupling these useful
tools to modern microscopic investigations.

96. OTHER APPLICATIONS
 Include-
-Resonance energy transfer,
-Stem cell research,
-Photobleaching studies,
-Lifetime imaging,
-Multiphoton microscopy,
-Total internal reflection,
-DNA hybridization,
-Membrane and ion probes,
-Bioluminescent proteins, and
-Epitope tagging.

97.  Confocal reflection microscopy can be utilized to gather
additional information from a specimen with relatively
little extra effort, since the technique requires minimum
specimen preparation and instrument re-configuration.
 In addition, information from unstained tissues is readily
available with confocal reflection microscopy, as is data
from tissues labeled with probes that reflect light.
 The method can also be utilized in combination with more
common classical fluorescence techniques.
 Examples of the latter application are detection of
unlabeled cells in a population of fluorescently labeled cells
and for imaging the interactions between fluorescently
labeled cells growing on opaque, patterned substrata

98. FLUORESCENCE MICROSCOPY

99. FLUORESCENT MICROSCOPY
 This method is used for demonstration of
naturally occurring fluorescent material and
other non-fluorescent substances or micro-
organisms after staining with some fluorescent
dyes e.g. Mycobacterium tuberculosis, amyloid,
lipids, elastic fibres etc.
 UV light is used for illumination.

100. •Uses UV light.
• Fluorescent
Substances absorb
UV light and emit
visible light.
• Cells may be
stained with
fluorescent dyes
(fluorochromes).
Fluorescence Microscopy

101. WHY FLUORESCENCE MICROSCOPY?
 In all types of microscopes, cell constituents are
not distinguishable, although staining dose, but
not totally.
 In fluorescent microscopy, various fluorescent
dyes are used which gives property of
fluorescence to only specific part of the cell and
hence it can be focused.
 Fluorescent microscopy depends upon
illumination of a substance with a specific
wavelength (UV region i.e. invisible region)
which then emits light at a lower wavelength
(visible region).

102. FLUORESCENCE PRINCIPLE
 When certain compounds are illuminated with
high energy light, they then emit light of a
different, lower frequency. This effect is known as
fluorescence.
 Often specimens show their own characteristic
autofluorescence image, based on their chemical
makeup.
 The key feature of fluorescence microscopy is that
it employs reflected rather than transmitted
light, which means transmitted light techniques
such as phase contrast and DIC can be
combined with fluorescence microscopy.

103. COMPONENTS
 Typical components of a fluorescence microscope
are:
 the light source (xenon arc lamp or mercury-
vapor lamp),
 the excitation filter,
 the dichroic mirror and
 the emission filter.

104. STAINING
 Many different fluorescent dyes can be used to
stain different structures or chemical compounds.
 One particularly powerful method is the
combination of antibodies coupled to a
fluorochrome as in immunostaining.
 Examples of commonly used fluorochromes are
fluorescein or rhodamine.

105. FUNCTIONING
 A component of interest in the specimen is
specifically labeled with a fluorescent molecule
called a fluorophore.
 The specimen is illuminated with light of a
specific wavelength (or wavelengths) which is
absorbed by the fluorophores, causing them to
emit longer wavelengths of light (of a different
color than the absorbed light).

106. APPLICATIONS
 Fluorescence microscopy is a critical tool for
academic and pharmaceutical research,
pathology, and clinical medicine.

107. The distance between the focal plane of the objective (f1) and the
focal plane of the eyepiece (f2) is called the tube length (l).
The object to be viewed is placed just outside the focal point at the
left side of the objective lens. The enlarged image is formed at a
distance l + f1 from the objective.

108. X-RAY MICROSCOPY

109. X-RAY MICROSCOPY
 less common,
 developed since the late 1940s,
 resolution of X-ray microscopy lies between that
of light microscopy and the electron microscopy.
 X-rays are a form of electromagnetic radiation
with a wavelength in the range of 10 to 0.01
nanometers, corresponding to frequencies in the
range 30 PHz to 30 EHz.

110. RECENT ADVANCES IN
MICROSCOPY

111. RECENT ADVANCES IN MICROSCOPY
 In the recent times, computers and chip
technology have helped in developing following
advances in microscopy.

112. IMAGE ANALYSERS AND MORPHOMETRY
 In these techniques, microscopes are attached to
video monitors and computers with dedicated
software systems.
 Microscopic images are converted into digital
images and various cellular parameters (e.g.
nuclear area, cell size etc) can be measured. This
quantitative measurement introduces objectivity
to microscopic analysis.

113. TELEPATHOLOGY (VIRTUAL MICROSCOPY)
 It is the examination of slides under microscope
set up at a distance. This can be done by using a
remote control device to move the stage of the
microscope or change the microscope field or
magnification called as robobic telepathology.
 Alternatively and more commonly, it can be used
by scanning the images and using the highspeed
internet server to transmit the images to another
station termed as static telepathology.
Telepathology is employed for consultation for
another expert opinion or for primary
examination

114. THANKYOU