MICROSCOPY

DR ASMA SULTANA
 Mention types of microscopes
• 1) Bright field microscope
• 2) Dark field microscope
• 3) Phase contrast microscope
• 4) Flourescent microscope
• 5) Electron microscope
• 6) Atomic force microscope
 WHAT IS SIMPLE MICROSCOPE
• Simple microscope is a magnifying glass
• Made of a simple convex lens with a short
focal length which magnifies the object
through angular magnification, thus producing
• An errect virtual image of the object near lens.
• It uses biconvex lens to magnify the image of a
• Specimen.
• Since it make use of one objective lens, its
• Magnifying capability is greatly limited.
 What is compound microscope.
• A compound microscope is an upright
microscope that uses two sets of lenses(a
compound lens system)to obtain a higher
• Magnification.it provides a two dimensional
image.Two sets of lenses are embedded-
objective lens and ocular(eye piece) lens.
• The total magnification of compound
microscope is the product of magnification of
• Objective lens and ocular lens.it has 2
• Adjustment screws- fine and coarse.
 What factors constitute magnification of
compound microscope
• The following are factors of magnification
• 1) focal length of objective lens
• 2) focal length of ocular lens.
 Wht is resolving power of microscope
• Resolving power of microscope is defined as the
reciprocal of the distance between two
• Objects which can be just resolved when seen
• Through microscope.
• Resolving power depends upon 1)wavelength.
• 2) refractive index of
medium between object and objective.
• 3)half angle of cone of light from one of object.
• How will you focus the given compound
• Microscope.
• A) to visualize a hanging drop preparation.wet
• Preparation of stool ?
• The microscope is adjusted for the reduced
• Light by lowering the condensor and using the
• Concave mirror. The edge of drop is focussed
• Under low power.The microscope is then
turned to high power to observe morphology
of bacteria.
 b) To visualize stained smears
• The microscope is adjusted for increased light
by raising the condensor and the slide is
examined with oil immersion objective using
the plane mirror.
• What is the function of oil when used with
• Oil immersion objective ?
• Oil immersion technique is used to increase the
• Resolving power of a microscope.
• The 100x lens immersed in a drop oif oil placed
• On a slide in order to eliminate any air gaps and
• loss of light due to refraction as the light passes
• From slide.
 Wht oils can be used for this purpose and
why ?
• 1)cedar wood oil
• 2)Liquid paraffin.
• Since it has same refractive index as that of
• Glass.
 Describe the principal and application of
Electron microscope ?
• An electron microscope uses accelarated
electrons as a source of illumination.because
the wavelength of electrons can be upto 1
lakh time shorter than that of visible light
photons and has much better resolving power
than light microscope
• Types-:
• 1)Transmission electron microscope:
• Specimen preparation: Specimen viewed
should be able to maintain its structure when It
is bombarded with electrons.hence only very
thin specimens( 20-100 nm) are suitable.
• To prepare thin specimens the steps followed
are- fixation,dehydration,embedding, slicing.
• Electron pathway-:
• Electrons generated by electron gun, which
travel in high speed.the medium should be fully
vaccum path,electrons can get deflected by
collisions with air molecules.
• 2) Scanning electron microscope-has resolution
of 7nm less.
 Application OF Electron microscope
• 1)VIRUS DETECTION:
• 1)Directly from clinical specimen
• 2)From tissue culture
• 3) After adding specific antiviral antibody to
specimen.
• 2)Contrast of EM:Negative staining with heavy
metals and shadowing.
3)freeze etching technique helps to disclose
• Shape of organelles within microorganisms.
 Describe principle and application of Dark
ground microscope.?
• Principle:
• In dark field(ground) microscope the object
appears bright against a dark background.this is
made possible by use of a special dark field
condensor.
• The dark field condensor has a central opaque area
that blocks light from entering objective lens
directly and has a peripheral annular hollow area
which allows light to pass through and focus on the
specimen obliquely.
• Only the light which is reflected by the
specimen enters the objective lens where as
the unreflected light does not enter the
objective.As a result the specimen is brightly
• Illuminated, but the background appears dark.
• APPLICATIONS:
• Dark field microscope is used to identify the
living unstained cells and thin bacteria like
spirocetes which can’t be visualised by light
microscope.
 Describe the principle and application of
phase contrast microscope.
• Principle:
The condensor is similar to that of dark field
microscope,consists of an opaque central area
with a thin tranparent ring which a hollow
cone of light.As this cone of light passes
through a cell, some light rays are bent due to
variation in density and refractive index with
the specimen and retarded by 1/4th of a
wavelength.
• The undeviated light rays strike a phase ring in
the phase plate while the deviated rays miss
the ring and pass through the rest of plate.
• The phase ring is constructed in such a way
that undeviated light passing through it is
advanced by a 1/4th of wavelegth.
• The deviated and undeviated waves will be half
wavelength out of the phase and will cancel
each other when they come together to form
an image.The background formed by
undeviated light is bright while unstained
object appears dark and well defined.
• Applications of phase contrast microscope:
• 1)to demonstrate microbial motility.
• 2) determining shape of living cells.
• 3) Detecting intracellular components such as
• Cell membrane,nuclei,mitochondria,spindles,
• chromosomes.,golgi apparatus,endospores
and inclusion bodies which become clearly
visible because they have refracive indices
markedly different from that of water.
 Describe principle and application of
fluorescent microscope.
• Principle:
• When fluorescent dyes are exposed to uv rays,
they become excited and are said to flouresce
ie, they convert this invisible short wavelength
rays into light of longer wavelength.
• Source of light may be a mercury lamp which
emits rays that pass through an excitation
filter.
• The excitation filter is so designed that it allows
only short wavelength uv light(about 400nm) to
pass through,blocking all other longer
wavelength rays.The exciting rays then get
reflected by a dichromatic mirror in such a way
that they fall on the specimen which is previously
stained by fluorescent dye and focussed under
microscope.The flourescent dye absorbs exciting
rays of short wavelength,gets activated and in
turn emits rays of longer wavelength.A barrier
filter positioned after objective lens removes any
uv light which would damage viewer’s eye.
 Applications -:
• Epifluorescence microscope-: simplest form of
microscope which has following applications:
• 1) Autoflouresence- cyclospora
• 2)Microbes coated with flourescent dyes-
• Acridine orange dye for detection of
plasmodium and filarial nematodes by a
method called Quantitative buffy coat(QBC).
• Auramine phenol to detect tubercle bacilli.
• 3) Immunofluorsence:
• To detect cell surface antigens or antibodies
bound to cell surface antigens.
• Two types:
• Direct and indirect.
• Confocal microscope-:
• 1) Advanced design of flouresence
microscope,uses point illumination and a pin
hole in an optically conjugate plane.
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