Basics of microscopy

Microscopy
• The examination of minute
objects by means of a microscope,
an instrument which provides an
enlarged image of an object not
visible with the naked eye.
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MICROSCOPE
• An optical instrument having a
magnifying lens or a
combination of lenses for
inspecting objects too small to
be seen or too small to be seen
distinctly and in detail by the
unaided eye. KKR1116 3

PARTS OF COMPOUND MICROSCOPE.
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PRINCIPLE
• The objective lens produces a magnified ‘real
image’ first image) of the object. This image
is again magnified by the ocular lens
(eyepiece) to obtain a magnified ‘virtual
image’ (final image), which can be seen by
eye through the eyepiece. As light passes
directly from the source to the eye through
the two lenses, the field of vision is brightly
illuminated.
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The resolving power of an objective lens is
measured by its ability to differentiate
two lines or points in an object. The
greater the resolving power, the smaller
the minimum distance between two lines
or points that can still be distinguished.
The larger the N.A., the higher the
resolving power
Resolving Power
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Numerical aperture.
• A mathematical constant that describes
the relative efficiency of a lens in bending
light rays.
• In general, the shorter the wavelength of
light being used, better the resolving
power.
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Magnification
• The degree of enlargement is the
magnification of the instrument.
• The magnification of an objective is
obtained as follows:
• Magnification =size of image/size of
object.
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Angular aperture.
• The angular aperture of an objective
lens is the angle between the most
divergent rays of the inverted cone of
light emerging from the condenser that
can enter the objective lens .rays cannot
enter the lens if their divergence from
the normal rays or optical axis is greater
than half the angular aperture.
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Focal length.
•The distance from a focal
point of a lens or mirror to
the corresponding principle
plane.
•Symbol: f
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Refractive index.
• Refractive index of a material is a
dimensionless number that describes how
fast light travels through the material .
• It is defined as:
n= c/v
c=speed of light in vacuum.
v=velocity of light.
Refractive index of water is 1.333
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Increasing the refractive index
corresponds to decreasing the speed
of light in the material.
It determines how much the path of
light is bent , or refracted when
entering a material.
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Focal point.
•The point at which the
light rays cross is called
the focal point f of the
lens.
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Types of microscope.
• Mainly two categories of microscopes, those in which the
light is used for observation.
• 1. light microscope
• a beam of light is used.
• It is a combination of optical lenses used for obtaining
magnification they are divided into following categories:
• a. bright field microscopy
• b . Dark field microscopy.
• c. fluorescence microscopy
• d. phase contrast microscopy.
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2. Electron microscope.
• A beam of electrons are used for
envisioning the object.it is divided into
four categories:
I. Transmission electron microscopy(TEM).
II. Scanning electron microscopy(SEM).
III. Scanning tunneling electron microscopy.
IV. Immunoelectron microscopy.
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Bright field microscopy.
• In bright field microscope, the specimen appears
as dark against the bright background. Stained,
fixed and live specimens are observed under a
bright field microscope. A bright-field microscope
is consists of A piece of apparatus, consisting of
an eyepiece, an objective lens, a condenser lens,
stage, and light source, which collects
electromagnetic radiation in the visible range
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Working Principle of Bright Field Microscope
• The specimen to be observed is placed on the stage of
a bright field microscope. The light will transmit through
the specimen from the source and then it will enter the
objective lens where a magnified image of specimen will
form. Then the light will enter an oracular lens or
eyepiece, where the image will further magnify a then
enter the into the user’s eyes. The viewers observe a dark
image against a bright background.
• In a bright-field microscope, only the scattered lights are
able to enter the objective lens and transmitted lights or
unscattered light rays are omitted, that’s why the viewer
sees a dark image against the bright field.
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Dark field microscopy.
• Dark-field microscopy is a technique that
can be used for the observation of living,
unstained cells and microorganisms. In
this microscopy, the specimen is brightly
illuminated while the background is dark.
It is one type of light microscopes, other
being bright-field, phase-contrast,
differential interface contrast, and
fluorescence. KKR1116 22

A dark field microscope is arranged so that the light source is
blocked off, causing light to scatter as it hits the specimen.
This is ideal for making objects with refractive values similar to the
background appear bright against a dark background.
When light hits an object, rays are scattered in all azimuths or
directions. The design of the dark field microscope is such that it
removes the dispersed light, or zeroth order, so that only the
scattered beams hit the sample.
The introduction of a condenser and/or stop below the stage
ensures that these light rays will hit the specimen at different
angles, rather than as a direct light source above/below the object.
The result is a “cone of light” where rays are diffracted, reflected
and/or refracted off the object, ultimately, allowing the individual
to view a specimen in dark field.
Principle of the Dark field Microscope
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Image formed by lenses.
• Lens are of two kind: convex lenses and concave lens.
• Convex lenses : This type of lens is thicker at the center
and thinner at the edges.
• An optical lens is generally made up of two spherical
surfaces. If those surfaces are bent outwards, the lens is
called a biconvex lens or simply convex lens. These types
of lenses can converge a beam of light coming from
outside and focus it to a point on the other side. This
point is known as the focus and the distance between the
center of the lens to the focus is called the focal length of
convex lens. However, if one of the surfaces is flat and the
other convex, then it is called a Plano-convex lens
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USES OF CONVEX LENSES.
These are used for a variety of purposes in our day-to-day lives. For
example,
•The lens in the human eyes is the prime example. So the most
common use of the lens is that it helps us to see.
•Another common example of the use of this type of lens is a
magnifying glass. When an object is placed in front of it at a distance
shorter than the focal length of the lens, it produces a magnified and
erect image of the object on the same side as the object itself.
•It is used to correct Hypermetropia or long-sightedness.
•It is used in cameras because it focuses light and produces a clear and
crisp image.
•More generally these are often used in compound lenses used in
various instruments such as magnifying devices like microscopes,
telescopes and camera lenses.
•A simple kind of these lenses can focus light into an image, but that
image won’t be of a high quality. For correcting the distortions and
aberrations, it is better to combine both types of lenses.
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Concave lenses.
• A concave lens is a type of lens that diverges a straight
light beam coming from the source to a diminished,
upright, virtual image is known as a concave lens. It can
form both real and virtual images. Concave lenses have at
least one surface curved inside. A concave lens is also
known as a diverging lens because they are shaped round
inwards at the center and bulges outwards through the
edges, making the light diverge on it. They are used for
the treatment of myopia as they make faraway objects
look smaller than they really are.
• The point in the lens where the light refracts and
diverges is known as the principal focus.
• The distance between the principal focus and the center
of the lens is known as the focal length.
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Uses of Concave Lens
Some uses of the concave lens are listed below:
Used in Telescope
Concave lenses are used in telescope and binoculars to magnify objects. As a convex lens
creates blurs and distortion, telescope and binocular manufacturers install concave lenses
before or in the eyepiece so that a person can focus more clearly.
Used in Eye Glasses
Concave lenses are most commonly used to correct myopia which is also called
nearsightedness. The eyeball of a person suffering from myopia is too long, and the images
of faraway objects fall short of the retina. Therefore, concave lenses are used in glasses
which correct the shortfall by spreading out the light rays before it reaches the eyeball. This
enables the person to see far away objects more clearly.
Used in Peepholes
Peepholes or door viewers are security devices that give a panoramic view if objects outside
walls or doors. A concave lens is used to minimize the proportions of the objects and gives a
wider view of the object or area
USES OF CONCAVE LENSES.
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Fold scope microscope.
• Foldscope is a paper microscope that is built by folding
the paper in origami fashion. Prof. Manu Prakash and Jim
Cybulski at Stanford University invented it during 2012.
• Unlike conventional microscope, foldscope does not
require electricity at all. Foldscope is light and simple
instrument that can be built in ultra-low- cost and is
simple to use making it accessible to more people across
the world. Today’s children of both developing and
developed countries, who hardly use the microscope
during the school life has been lucky enough to see the
biggest revolution in the field of science through the use
of paper microscope
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Construction.
• It is assembled from a punched sheet of
cardstock , a spherical glass lens , a light
emitting diode and a diffuser panel.
Along with a watch battery that power
the led , foldscope is about the size of a
bookmark.it weights 8 grams and comes
in a kit with multiple lenses that provide
magnification from 140×2,000x.
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Uses of foldscope microscope.
• Plant pathologist used it for examining banana crops
and maasai children in tanzala who used it to check
cow feces for parasites
• Used to observe microbes in local environment.
• Used as a teaching tool for students in biology,
chemistry and physics.
• Pollen of pithecellobium dulce is seen with the
foldscope microscope.
• Pollen of the delonix regia ,samanea. A.niger ,
fungus on garlic cloves.
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Light microscope principle.
• The light microscope is an instrument for
visualizing fine detail of an object. It does
this by creating a magnified image
through the use of a series of glass
lenses, which first focus a beam
of light onto or through an object, and
convex objective lenses to enlarge the
image formed
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Electron microscope principle.
• Electron microscopes use signals arising from the interaction of an electron beam with the sample
to obtain information about structure, morphology, and composition.
• The electron gun generates electrons.
• Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight
beam.
• To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is
applied between tungsten filament and anode.
• The specimen to be examined is made extremely thin, at least 200 times thinner than those used in
the optical microscope. Ultra-thin sections of 20-100 nm are cut which is already placed on the
specimen holder.
• The electronic beam passes through the specimen and electrons are scattered depending upon the
thickness or refractive index of different parts of the specimen.
• The denser regions in the specimen scatter more electrons and therefore appear darker in the
image since fewer electrons strike that area of the screen. In contrast, transparent regions are
brighter.
• The electron beam coming out of the specimen passes to the objective lens, which has high power
and forms the intermediate magnified image.
• The ocular lenses then produce the final further magnified image.
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References.
• At the bench – a laboratory navigator by: Kathy Becker.
• Textbook of microbiology for degree students
By: sullia and shantaram.
• General microbiology by: chandrakant kelmani.
• Textbook of microbiology by :pleczar and chain
• microbiology by: power and diagniwala.
• General microbiology by: roger Y.stainier
John L.ingraham.
Mark l. wheels
Page R. painter.
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Basics of microscopy

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Basics of microscopy