Research is "creative and systematic work undertaken to increase the stock of knowledge". It involves the collection, organization and analysis of evidence to increase understanding of a topic, characterized by a particular attentiveness to controlling sources of bias and error.

1. MICROSCOPE
Lecture 2
ll

2. What is microscope?
A microscope is an instrument used to see objects
that are too small for the naked eye.

3. Properties of a microscope
A good microscope should have at least three
properties:
• Good Resolution
• Good Contrast
• Good Magnification

4. Good Resolution :-
• Resolution power refers to the ability to produce
separate images of closely placed objects.
• So that they can be distinguished as two separate
entities.
The resolution power of-
• Unaided human eye is about 0.2mm(200 µm)
• Light microscope is about 0.2 µm.
• Electron microscope is about 0.5 nm.
Immersion Oil has a higher refractive index than air,
hence, use of oil enhances the resolution
power of a microscope.

5. Good Contrast:
• Contrast is improved by staining the specimen.
• When the stain bind to the cells, the contrast is
increased.

6. Good Magnification:
• Ocular lens with a magnification power of
10X.
• Objective lens-
• Scanning (4X)
• Low power (10X)
• High power (40X)
• Oil immersion (100X)

7. Total magnification of a field is the product of
the magnification of objective and ocular lens:
• Scanning field (40X)
• Low power field (100X)
• High power field (400X)
• Oil immersion field (1000X)

8. TYPES OF MICROSCOPE
The following types of microscopes are in use
now-
• Bright field or light microscope
• Dark field microscope
• Phase contrast microscope
• Fluorescence microscope
• Electron microscope

9. Bright field or light microscope
• Light microscope forms a dark image against a
brighter background, hence the name bright
field.
Structure:
• The parts of light microscope is divided into
three groups-
• Mechanical part
• Magnifying part
• Illuminating part

10. Mechanical part:
• Base: It holds various part of microscope, such as
the light source, the fine and coarse adjustment
knobs.
• C- shaped arm: It hold the microscope, and it
connects the ocular lens to the objective lens.
• Mechanical stage: The arm bears a stage with
stage clips to hold the slides and the stage control
knobs to move the slide during viewing.
• It has an aperture at the center that
permit light to reach the object from
the bottom.

11. Magnifying part:
• Ocular lens: The arm contains an eye piece that
bears an ocular lens of 10X magnification power.
• Microscope with two eye pieces are called as
binocular microscopes.
• Objective lens: The arm also contains a revolving
nose piece that bears three to five objectives with
lenses of differing magnifying power
(X,10X,40X, and 100X)

12. Illuminating parts:
• Condenser: It is mounted beneath the stage
which focuses a cone of light on the slide.
• Iris diaphragm: it control the light pass
through the condenser.
• Light source: it may be a mirror or an electric
bulb.
• Fine and coarse adjustment knob: They
sharpen the image.

13. MICROSCOPE

14. Principle :
• The rays emitted from the light source pass
through the iris diaphragm and fall on the
specimen.
• The rays passing through the specimen is
gathered by the objective and a magnified image
is formed.
• This image is further magnified by the ocular
lens to produce the final magnified virtual
image.

15. Compound microscope Dark-field microscope

16. Dark field microscope:
• In dark field microscope, the object appears bright
against a dark background.
• It is made possible by special dark field condenser.
• Hence the name dark field microscope.
Principle:
• The dark field condenser has a central opaque area
that blocks light from entering the object lens
directly and has a peripheral annular hollow area
which allows the light to pass through and
focus on the specimen obliquely.

17. • Only the which is reflected by the specimen enters
the objective lens whereas the unreflected light
does not enter the objective.
• As a result, the specimen is brightly illuminated;
but the background appears dark.
Application:
• Dark field microscope is used to identify the living,
unstained cells and thin bacteria like spirochetes
which is can not visualised by light microscope

18. Phase contrast microscope:
• As per name, in phase contrast microscope the
contrast is enhanced .
• This microscope visualizes the unstained living
cells by creating difference in contrast between
the cells and water.
• It converts slight differences in refractive index
and cell density into easily detectable variations
in light intensity.
• Contrast can be enhanced by staining of the
specimens, but as staining kills the
microbes, the properties of living cells
cannot be studied.

19. Principle:
• The condenser is similar to that of dark field
microscope, consist of an opaque central area with
a thin transparent ring, which produces a hollow
cone of light.
• As this cone of light passes through a cell, some
light rays are bent due to variations in density and
refractive index within the specimen and are
retarded by about one fourth of a wave length.
• The undeviated light rays strike a phase
plate,(a special disk located in the objective)
while the deviated rays miss the ring and pass
through the rest of the plate.

20. • The phase ring is constructed in such a way that
the undeviated light passing through it is
advanced by one-fourth of a wavelength out of the
phase and will be cancel each other when they
come together to form an image.
• The background, formed by undeviated light ,is
bright ,while the unstained object appears dark
and well defined.
Summary of application-
• The light rays go throughcondenser
specimen phase ring objective lens
ocular lens

21. Ray diagram of Phase contrast microscope

22. Application:
• To study unstained living cells
• Detailed examination of internal structures in
living microorganisms.
• To study flagellar movements and motility of
bacteria and protozoans.
• To study intestinal and other live protozoa such
as amoebae and Trichomonas.
• To examine fungi grown in culture.

23. Fluorescence microscope:
Refers to any microscope that uses fluorescence property
to generate an image.
Principle:
• When fluorescent dyes are exposed to ultraviolet rays,
they become excited and are said to fluoresce, i.e. they
convert this invisible, short wavelength rays into light
of longer wavelengths (i.e. visible light).

24. Ray diagram of Fluorescent Microscope

25. • The 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,
called as the exciting wavelength of light) to pass
through; blocking all other long wavelength rays.

26. • The exciting rays then get reflected by a
dichromatic mirror in such a way that they fall on
the specimen which is priorly stained with
fluorescent dye.
• The fluorescent dye absorb the the exciting
rays of short wavelength, gets activated and
it turns emits fluorescent rays of higher
wavelength.

27. • The barrier filter positioned after the objective
lenses removes any remaining UV light, which
could damage the viewer eyes, or blue and violet
light, which would reduce the image contrast.
Applications:
Epifluorescence microscope:
It is the simplest form of fluorescence microscope,
which has the following applications.
• Auto fluorescence: Some microbes directly
fluoresce when placed under UV lamp e.g.
Cyclospora.

28. Microbe coated with fluorescent dyes:
Certain microbes fluoresce when they are stained
with fluorochrome dyes. e.g.
• Acridine orange dye is used in QBC examination
for the detection of malarial parasites.
• Auramine phenol is used for the detection of
tubercle bacilli.
Immunofluorescece:
• It uses florescent dye tagged immunoglobulin
to detect cell surface antigen or antibodies
bound to cell surface antigens.

29. Electron Microscopy :
• An Electron microscope uses accelerated
electrons as a source of illumination.
• Because the wave length of electrons can be up
to 100,000 time shorter than that of visible light
photons.
• The electron microscope has a much better
resolving power than a light microscope.
• Hence, it can reveal the details of flagella,
fimbriae and intracellular structures of a cell.

30. • It was invented by German Physicist Ernst and
Ruska in 1931.
Difference between light microscope and electron microscope

31. Electron microscope are of two types:
• Transmission electron microscope
• Scanning electron microscope
Transmission electron microscope
• Electrons are generated by electron gun, which
travel in high speed.
• The medium of travel in EM should be a fully
vacuum path because in air path, electrons can get
deflected by collisions with air molecules.

32. Electron pathway:
• Electron pass through a magnetic condenser and then
bombardon thin sliced specimen mounted on the
copper slide.
• The specimen scatters electrons passing through it,
and then the electron beam is focused by magnetic
lenses to form an enlarged, visible image of the
specimen on a fluorescent screen.
Measures to increase contrast of EM –
Staining:
In electron microscope the stain is used are solution of
heavy metal salts like lead citrate and uranyl acetate.

33. Negative staining:
The specimen is spread out in a thin film with
heavy metals like phosphotungstic acid or uranyl
acetate.
Shadowing:
This technique is particularly useful in studying
virus morphology, bacterial flegella, and
plasmids.

34. Scanning electron microscope
• Scanning electron microscope has been used to
examine the surface of microorganisns in great
detail.
• It has a resolution of 7nm or less.
• The SEM differs from TEM, in producing an
image from electrons emitted by an object’s
surface rather than from transmitted electrons

35. Rules of using a microscope
• Always carry with 2 hands
• Only use lens paper for cleaning
• Do not force knobs
• Always store covered
• Be careful of the cords