2. Microscope
A microscope is an instrument used to see objects that are too small to be
seen by the naked eye. Microscopy is the science of investigating small
objects and structures using such an instrument. Microscopic means
invisible to the eye unless aided by a microscope.
There are many types of microscopes, and they may be grouped in different
ways. One way is to describe the way the instruments interact with a sample
to create images, either by sending a beam of light or electrons to a sample
in its optical path.
The most common microscope is the optical microscope, which uses light to
pass through a sample to produce an image.
The major types of microscopes are the Compound Microscope, Phase
contrast microscope, fluorescence microscope, the electron microscope (the
transmission electron microscope and the scanning electron microscope).
3. When a ray of light passes from one medium to another, refraction
occurs—that is, the ray is bent.
The refractive index can be seen as the factor by which the speed
and the wavelength of the radiation are reduced.
Our eyes cannot focus on objects which are two small. This
limitation may be overcome by using a convex lens as a simple
magnifier (or microscope) and holding it close to an object.
A magnifying glass provides a clear image at much closer range, and
the object appears larger.
Resolving power: Ability two differentiate two particles which are
very close.
The human eye has resolving power 0.25 mm.
4. Convex lens:
A Convex lenses are thicker at the middle. Rays of light that
pass through the lens are brought closer together. A convex
lens is a converging lens.
When parallel rays of light pass through a convex lens the
refracted rays converge at one point .
The distance between the principal focus and the centre of
the lens is called the focal length.
A magnifying glass is a convex lens which produces a
magnified image of an object.
• Convex lens used in microscopes and
telescopes for magnifying the objects.
5. Concave lens:
Concave lenses are thinner at the middle. Rays of light that pass through the
lens are spread out (they diverge). A concave lens is a diverging lens.
The image formed by this lenses is smaller.
7. CIA Component Date
CIA:1 Malaria?
Malaria history? Outbreak of Malaria
in the world and in India?
Statistical information on deaths cases
in India?
Research Studies on Malaria?
Immune response, Transmission,
Diagnosis, Prevention
And Vaccination?
Submission
on before
28/06/2017
1740601 to
1740620
8. 2 Write key points on HIV?
HIV virus pandemic history? Outbreak of
HIV in the world and in Indian?
Statistical information on deaths cases in
India?
Research Studies on HIV virus?
Immune response, Transmission, Diagnosis,
Prevention
And Vaccination?
17406021-
17406040
3 Write key points on Hepatitis B Virus?
Research Studies on Hepatitis B Virus?
Hepatitis B Vaccine, Symptoms, Treatment,
Cure & Transmission?
17406041-
17406060
9. 4 Write key points on Tuberculosis?
Research Studies on Tuberculosis?
Statistical information on deaths cases in
India?
Tuberculosis, Symptoms, Treatment, Cure
& Transmission?
17406061-
17406067
10. 16/06/2017
Bright field microscope or Light microscope or compound
microscope:
A light source, either a mirror or an electric illuminator, is
located in the base.
Mirror: The mirror reflects light which is transmitted through
the object. The mirror has two planes one is concave other is
plane. When natural light is available the plane mirror used
for reflection.
Diaphragm: this is used for control the amount of light
transmitted through the object.
11. The substage condenser: is mounted within or
beneath the stage.
it consists of convex lenses which concentrate and
intensify the light reflected by the mirror and produce
a cone of light on the slide. The generally used
condensers are Abbe condensers. Its position often is
fixed in simpler microscopes but can be adjusted
vertically in more advanced models.
Stage: The object to be observed is kept on a glass slide
and placed on the stage. It may have clips to keep the
slide in desired position.
12. The curved upper part of the microscope holds the body tube, to
which a nosepiece and one or more eyepieces or oculars and
objective lens are attached.
More advanced microscopes have eyepieces for both eyes and
are called binocular microscopes.
Lenses: The eyepiece with different magnification 5-20 times, it
has field lens toward the objective and eye lens close to observers
eye.
The objective lens with three different magnifications, i.e: low
power (10X), high power (40-45x) and oil immersion (90-100 X).
The objective lens are mounted on a revolving nosepiece for
convenience.
The eyepiece and objective lens are fitted at the two ends of
hollow tube called Body tube.
13. The objective lens forms an enlarged real image within the
microscope, and the eyepiece lens further magnifies this
primary image. When one looks into a microscope, the
enlarged specimen image, called the virtual image, appears to
lie just beyond the stage about 25 cm away.
The total magnification is calculated by multiplying the
objective and eyepiece magnifications together. For example,
if a 45x objective is used with a 10x eyepiece, the overall
magnification of the specimen will be 450x.
If a 100x objective used with 10x eyepiece the magnification of
the specimen will be 1000x.
14. Microscope Resolution
The most important part of the microscope is the objective
lens, which must produce a clear image, not just a
magnified one. Thus resolution is extremely important.
Resolution is the ability of a lens to separate or distinguish
between small objects that are close together.
The major factor in resolution is the wavelength of light
used. The wavelength must be shorter than the distance
between two objects otherwise the objects will not be seen
clearly.
Thus the greatest resolution is obtained with light of the
shortest wavelength.
15. Phase-Contrast Microscope:
• This microscope developed by Fritz Zernikes a Dutch scientist ,
who was awarded Nobel prize in 1953 for this contribution.
• Un-pigmented living cells are not clearly visible in the bright
field microscope because there is little difference in contrast
(vision of color) between the cells and water.
• Thus microorganisms often must be fixed and stained before
observation to increase contrast and create variations in color
between cell structures.
• Uses: Phase-Contrast Microscope used to study unstained
structures if microbial cells and internal components of bacteria
such as endospores and granules etc.
• It is used to study the osmotic behaviour of living cells and plant
and bacterial cell division.
16.
17. • A phase-contrast microscope converts slight differences in
refractive index and cell density into easily detected variations
in light intensity.
• The phase-contrast microscope has an annular diaphragm and
condenser which produces a hollow cone of light.
• As this cone passes through a cell, some light rays are bent
(deviated) due to variations in density and refractive index
within the specimen.
• Un-deviated light rays strike a phase ring in the phase plate (a
special optical disk located in the objective) while the deviated
rays miss the ring and pass through the rest of the plate.
18. • The phase ring changes the wave length of
deviated rays and un-deviated rays.
• The background, formed by un-deviated light,
is bright, while the unstained object appears
dark and well-defined.
• This type of microscopy is called dark-phase-
contrast microscopy. Color filters often are
used to improve the image.
19. The Fluorescence Microscope
• Fluorescence Microscope developed by Coons in 1945, in which
the specimen stained with fluorescent dye.
• The fluorescent substance absorbs light of one wave length and
emits light at different wave length.
• For example Fluorescein isothiocynate absorb blue light and
emits green light.
20. • The fluorescence microscope exposes a specimen to ultraviolet,
or blue light .
• A mercury vapor lamp is a source of light produces an intense
beam.
• The light passes through an exciter filter that transmits only the
desired wavelength (short wave length).
• A darkfield condenser provides a black background against
which the fluorescent objects glow brightly .
.
21. • Usually the specimens have been stained with dye molecules,
called fluorochromes. Upon exposure to light of a specific
wavelength that appear brightly .
• The microscope forms an image of the fluorochrome-labeled
microorganisms.
• A barrier filter positioned after the objective lenses removes
any remaining ultraviolet light, which could damage the
viewer’s eyes, or blue and violet light, which would reduce the
image’s contrast.
23. • The fluorescence microscope has become an essential tool in
medical microbiology and microbial ecology.
• Bacterial pathogens (e.g., Mycobacterium tuberculosis, the cause
of tuberculosis) can be identified after staining them with
fluorochromes.
• The fluorescence microscope is used to observe microorganisms
stained with fluorochromes such as acridine orange and DAPI
(diamidino-2- phenylindole, a DNA-specific stain).
24. a). A mixture of Micrococcus luteus and Bacillus subtilis (the rods). The live bacteria
fluoresce green; dead bacteria are red.
26. Electron microscope
EM was invented by Knoll and Ruska in 1932.
An electron microscope is a microscope that uses a beam of
electrons as a source of illumination rather than the beam
of light.
As the wavelength of an electron can be up to 1,00,000
times shorter than that of visible light photons.
Electron microscopes have a higher resolving power and
magnification than light microscopes and can reveal the
structure of smaller objects.
27. • The 100 Kilo volts voltage used in electron microscope, the
wave length of the electron beam is approximately 0.005nm
and providing resolution of approximately 0.2 nm.
• This resolution is roughly 1000 times better than that of the
light microscope and magnification of electron microscope is
1,00,000x.
• Therefore an electron microscope provides sufficient
magnification and resolution to view viruses and the internal
structures of all microorganisms.
• The living cells cannot be examined because cannot survive the
destructive action of the stream of electrons.
28. • Electron microscope use electromagnetic lenses rather than
glass lenses in light microscope.
• Instead of producing the image on the retina of the eye, it is
formed on a fluorescent screen or a photographic plate.
• Instead of mounting the specimen on a glass slide, it is held
on a copper grid that allows electrons to pass through the
specimen.
Copper grid
29. Characteristic Light microscope Electron
Microscope
High magnification 1000-1500x 1,00,000x
Best resolution 0.2µm 0.5nm
Radiation source Visible light Electron beam
Type of lens Glass Electromagnetic
Source of contrast Differential light
absorption
Scattering of
electrons
Specimen mount Glass slide Copper grid
Focusing
mechanism
Adjustment of lens
position
Adjustment of
current to the
magnetic lens
30. Two types of electron microscopes:
1.Transmission electron microscope (TEM)
2.Scanning electron microscope (SEM)
31. The Transmission Electron Microscope
• The electron gun generates an electron beam from a thin
tungsten filament.
• The electron beam is focused on the specimen with an
electromagnetic condenser lens.
• Air is removed form the column containing the lenses and
specimen by high efficiency Vacuum system.
• This done so as to obtain a clear image since electrons are
deflected by collisions with air molecules.
• The specimen scatters electrons when magnetic lenses
focused a beam of electrons on to the specimen.
32. Dense regions in the specimen
scatter more electrons and therefore
appear dark image, whereas in thin
regions are brighter.
A denser region in the specimen
scatters more electrons as a result
fewer electrons strike that area of
the screen.
The enlarged image of specimen
visible on a fluorescent screen.
The image on the screen can also
photographed for permanent record.
The photograph called as
Transmission electron micrograph.
33.
34. Summary:
• A heated tungsten filament in the electron gun generates a beam of
electrons that is then focused on the specimen by the condenser lenses.
• Since electrons cannot pass through a glass lens, magnetic lenses are used
to focus the beam.
• The column containing the lenses and specimen must be under high
vacuum to obtain a clear image because electrons are deflected by
collisions with air molecules.
• A denser region in the specimen scatters more electrons and therefore
appears darker in the image since fewer electrons strike that area of the
screen.
• In contrast, electron-transparent regions are brighter. The screen can also
be moved aside and the image captured on photographic film as a
permanent record.
35. The Ultra thin sections of specimen are prepared by freezing in
liquid nitrogen and sectioning with a diamond of glass knife.
The sections are floated in water and picked up on a copper
grid and inserted into the vacuum chamber of the microscope.
The electron beam is focussed on the section and manipulated
by magnetic lenses.
38. Scanning electron microscope (SEM)
• Scanning electron microscope develop in 1960s used to examine
the surface of microorganisms.
• SEM gives three dimensional appearance of the specimen
surface.
• SEM is very useful in studying the surface of bacteria cells,
fungi, protozoa and viruses.
• SEM allows only surface of specimen in their natural state
without staining.
39. The specimen is put into the vacuum chamber and covered with
a thin coating of gold or platinum.
Coating prevents the penetration of electrons on surface of
specimen and sharpens the image.
SEM produce an image from electrons emitted or scattered by
an object surface rather than form transmitted electrons.
40. It consists of electron gun which produces a beam of electrons
called primary electron beam.
These electrons pass through electromagnetic lenses and strikes
the surface of the specimen.
When the beam of electrons strikes the specimen secondary
electrons are released form specimen surface.
The secondary electrons are transmitted to electron collector or
detector.
41. The secondary electrons are collected by detector and used
to generate a signal that is processed electronically.
The processed signals produce an image on cathode ray
tube screen or photographic place.
The signal is sent to a cathode-ray tube and produces an
image like a television picture, which can be viewed or
photographed.
