Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

1. MICROSCOPY
SHRI SHANKARACHARYA MAHAVIDYALAYA
JUNWANI, BHILAI.
Dr. RACHNA CHOUDHARY
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2. MICROSCOPY
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• INTRODUCTION
• HISTORY
• PRINCIPLE
• FACTORS OF MICROSCOPY
• TYPES OF MICROSCOPY
 LIGHT MICROSCOPY
A. BRIGHT-FIELD MICROSCOPY
B. DARK-FIELD MICROSCOPY
C. FLUORESCENCE MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
 ELECTRON MICROSCOPY
A. TRANSMISSION MICROSCOPY
B. SCANNING MICROSCOPY
• APPLICATIONS
• CONCLUSION
• REFERENCE
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3. MICROSCOPY
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• Microscopy is the science which deals “with the use of microscope
instrument that magnifies the size of the object & interpretation of
their magnified images”.
• Microscope is the technology of making very small things visible to
the human eye. Therefore, microscope is a major tool of the
microbiologist & biotechnologist.
• At the magnification of 1,000x most of the microorganisms, e.g.:-
Fungi, Bacteria, Mycoplasma, algae, & protozoa can be viewed &
this can be achieved with a light microscope.
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4. MICROSCOPY
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• Antony Van Leeuwenhoek (1676)
discovered the microbial world through
the use of a single microscope containing
a single biconvex lens of shorter focal
length.
• Robert Hooke point the ‘cell’ built
microscopes with two lenses called
compound microscope.
• The Dutch spectacle-maker, Zaccharias
Jansen, is also credited with the
development of compound light
microscope.
• In 1830, many improvements were made
by Joseph Lister which resulted in the
development of many types of
microscopes that are being used now a
days.
Zacharias Jansen
1588-1631
Anthony van Leeuwenhoek
1632-1723
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5. MICROSCOPY
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• Microscopy is the most important instrument of any biological
laboratory. Used to magnifying the size of the object.
• By the help of resolution provided by this instrument very small
organisms or substances are magnified and made visible through
the naked eyes.
• Thus, microscopy is the major tool for microbiologist &
biotechnologist.
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6. MICROSCOPY
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1. Magnification
• The primary of a microscope is magnification that is the power of
enlargement of image of an object.
• Its is the ratio of size of image to the size of object.
Image distance
Magnification = --------------------
object distance
• Three types of objective lens of different magnification are used:
a. Low power- 10x
b. High power- 45x
c. Oil emulsion- 100x
2. Contrast
• It refers to difference in light intensity in order to the perceived to the
microscope, an object must possess a certain degree of contrast with its
surrounding medium.
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3. Resolution
• The resolving power of a microscope is the to distinguish two adjacent
point as separate and distinct rather then a blurred image.
• The greater the resolving power of the microscope the more detailed
can be see in the specimen.
• Resolving power of microscope is determined by three factors:
i. Size of the objective lens.
ii. Wavelength of light passing through the specimen.
iii. The refractive index of the material between the objective lens
and the specimen.
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8. MICROSCOPY
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1. LIGHT MICROSCOPY
A. Bright-Field light microscopy
B. Dark-Field light microscopy
C. Fluorescence light microscopy
D. Phase-contrast light microscopy
2. ELECTRON MICROSCOPY
A. Transmission electron microscopy
B. Scanning electron microscopy
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9. MICROSCOPY
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 Light as source of
illumination
 Glass lenses
 Limited resolution
(loses resolving
power at
magnifications
above 2000X
LIGHT MICROSCOPY
Fig-1 light microscope
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10. MICROSCOPY
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A. BRIGHT FIELD MICROSCOPY
PRINCIPLE
• Bright field use the visible light as the source of illumination.
• Light microscope with a single lens are called simple microscope.
• Compound microscope with the two lens system the objective lens place near specimen
and the occular lens or eye piece located next to the eye.
Basic part of Bright-field microscope
 A broad base
 Curved arm
 Adjustable light source or mirror
 Fine and core adjustable nose
 Body tube
 Stage or platform
 Diaphragm
 Condenser
 Ocular lens
 Eye piece
 There are three types of eye piece
a) Huggensian eye piece
b) Hyper –place eye piece
c) Compensating eye piece
Fig-2 bright-field microscopy
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11. MICROSCOPY
 The object to be viewed with the compound light microscope is normally
placed on a glass slide and illuminated with a light source. The specimen is
focused by moving the ocular lens & objective lens together relative to the
specimen until the image is clear.
 When the specimen has been focused the objective lens magnifies the
specimen & produces earlier image.
 The real image is projected to the microscope to the ocular lens which
magnifies the real image and produces an image seen by the observer and
called as virtual image.
 The resolving power of the lens system is important in microscopy because it
indicates the size of the smallest object that can be seen clearly.
 Resolving power varies for each objective lens and depends on-
1. Wavelength of light used in a optical system,
2. Numerical aperture.
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Conti..A. BRIGHT FIELD MICROSCOPY
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12. MICROSCOPY
A. BRIGHT FIELD MICROSCOPY
NUMERICALAPPARATURE
 It is the light gathering capacity of the objective lens.
 It is a measurement of the angle of maximum cone of light that enter the objective.
NA = n Sin𝜽
where, N = refractive index of medium
sin 𝜃 = one and half angle created by light passing through condenser and specimen
& transmitted to object.
o In case of dry air
NA= 1
o In case of oil medium
NA= 1.33
 The greater the NA the greater is the resolving power.
LIMIT OF RESOLUTION
 The smallest distance by which two points can be separated & distinguished as two separate
objects.
Resolution = 𝝀
-------
2NA
 Higher resolution obtained with shorter wavelength & maximum NA.
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13. MICROSCOPY
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B. DARK-FIELDMICROSCOPY
 In dark field microscopy the background
remains dark & only the objects
illuminated. It is opposite to that of
Bright-field microscopy in which
specimen appears darker against light
background.
 Dark-field microscopy operates on the
principle of scattering which means a ray
of light changes direction or scatters
when it strikes & bounces off a small
object.
 In this a special kind of condenser with an
opaque disc or “dark field stop” is
provided. Thus, the light rays reach the
object in the form of the hollow cone.
Fig-3 dark-field microscopy
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14. MICROSCOPY
B. DARK-FIELD MICROSCOPY
 The disc block the light that could enter the objective directly & redirects the light
beam so that it goes to the specimen but misses the objective lens.
 The only light rays that enters objective lens & reach the eyes are those that have been
scattered by striking the specimen.
 In tis way specimens appears bright against a dark microscopic field.
APPLICATION
 Helps in examining movement of motile cells, live microorganisms that are
either invisible in the ordinary light microscope
 In diagnostic of microorganisms.
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15. MICROSCOPY
C. FLUORESCENCE MICROSCOPY
 The fluorescence microscopy differs from the Bright-field microscopy as
it uses a mercury vapour arc lamp or a halogen instead of the
incandescent lamp.
PRINCIPLE
 The principle of fluorescence microscopy is a diagnostic technique
called the fluorescent-antibody. Antibodies are natural defense
molecule that are produced by humans and many animals in
reaction to a foreign substance or antigen.
TYPES OF FLUORESCENCE
1. Auto fluorescence - collagen fibers (blue green light).
1. Secondary fluorescence - commonly used dye Congo red, eosin.
1. Induced fluorescence – some substances on treatment with some
chemical shows fluorescence.
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16. MICROSCOPY
COMPONENTS &
INSTRUMENTATION
1. LIGHT SOURCE – Mercury arc lamp,
ultraviolet light of shorter
wavelength.
2. HEAT FILTER – Removes infrared
rays.
3. EXITER FILTER – Allow only
required wavelength to pass through
and block others.
4. DICHRONIC MIRROR – Divide &
divert the beam, reflect light of
certain wavelength but transmit other.
5. CONDENSOR – Dark field condenser
is provide black background against
which the fluorescent object glow.
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Fig-3 fluorescence microscopy
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17. MICROSCOPY
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C. FLUORESCENCE MICROSCOPY
6. BARRIER FILTER – Remove exited wavelength
APPLICATION
 Detection various material e.g. protein can be detected by staining
with rod amine.
 Banding pattern of chromosome.
 Fluorescent antibody technique or Immuno-fluorescent.
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18. MICROSCOPY
D. PHASE CONTRAST MICROSCOPY
• Phase-contrast microscopy is based on the principle that rays of light
move at different speed through materials of different refractive index.
• The phase contrast microscope amplifies the slight difference in
refractive index of the cell and that of its aqueous environment and
converts it to a difference in contrast.
• The phase-contrast microscope consists of special condensers and
objectives that enable one to increase the contrast between the
transparent components in the cell by exploiting differences in their
densities.
 PRINCIPLE
 Phase contrast microscopy is used for studying living cell to
convert the invisible small phase changes caused by cell
component into visible intensity changes.
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19. MICROSCOPY
D. PHASE-CONTRAST MICROSOPY
 When light rays passed through the living
cells they undergo phase changes due to
different refractive indices & thickness of
cell organelles
 When light rays are passed through cell
organelle, they are transmitted at a
velocity inversely proportional to
refractive index of the cell organelle. Cell
organelle are of different refractive
indices.
 The light rays emerging would show
variable phase changes.
 This invisible phase changes re converted
into visible intensity changes by phase-
contrast microscopy.
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Fig4-phase-contrast microscopy
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20. MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
 The more the refractive index & thickness the more will be the
change in the phase.
 The cells and their component show phase changes value of
phase change is one- fourth of light. This phase change is
imperceptible to the human eye.
 The principle behind the phase-contrast microscopy is to convert
the imperceptible phase change.
CONSTRUCTION
 It is specially designed light microscope with annular
diaphragm and annular phase plate fitted into it.
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21. MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
 They unable to increase the contrast between the transparent into
the ell by exploiting differences in their density.
 Annular diaphragm or annular stock is a disc with a thin
transparent wing at a lower focal plane of the condenser.
 It consist of a circular disc with a circular grove through where
light rays are allowed to pass.
WORKING
 Light rays pass through the annular group of the annular
diaphragm.
 This rays are focused on the on the object.
 From the object two types of rays immersed out. one is refracted
rays which under goes a phase change. And other is central rays
which does not under goes any phase –change.
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22. MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
 The refracted rays are band due to refraction in density and refractive
index with in the specimen and get refracted by about one-fourth
wavelength.
IMAGE FORMATION
 Depending on the type of phase plate used image formation takes
place is of two types-
Image formation by positive contrast
• Formed by subtractive super position of central & diffracted rays.
• The object appears dark against the light background also called as
dark positive contrast.
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D. PHASE-CONTRAST MICROSCOPY
Image formation by negative contrast
• Formed by the super position of the central & diffracted rays.
• The specimen appear bright against dark background that may
called as bright phase contrast.
• In this negative plate is used.
ADVANTAGES
 We can see living cells and there is no need for staining.
 Highly transparent material can be seen.
 Intracellular component can be observed, e.g. endospores.
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24. MICROSCOPY
ELECRTON MICROSCOPY
 The electron microscope is an optical instrument which utilizes electrons as
a source of illumination for observing objects at a great magnification.
 It can achieve a very high power of resolution because it uses electrons of
much shorter wavelength.
 Use electromagnetic lenses to focus a beam of electrons onto a
specimen.
 This required 10,000x plus magnification.
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26. MICROSCOPY
TRANSMISSION ELECTRON MICROSCOPY
 PRINCIPLE
 Higher magnification and higher resolution.
 The wavelength of electron one lakh time shorter then that of light
rays.
 Image is produced on fluorescence on photographic plate.
 Instead of mounting the specimen on a glass slide it held on a
proper way.
 Instead of using light to that absorb light, to increase contrast
tungsten which absorbs electron are used.
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27. MICROSCOPY
CONSTRUCTION
1. ELECTRON GUN
 It consist of a hot tungsten filament. It is the source
of electrons forming the beam.
2. ELECTROMAGNETIC LENS
 The electromagnetic lens corresponds to the
condenser, objective lens and ocular lens.
3. MICROSCOPE COLUMN
 It consist of an evacuated metal tube.
4. FLUORESCENCE SCREEN
 Since electron are harmful to our eyes magnified
image observed from fluorescence screen.
5. VACUUM PUMP
 Electron are reflected by collision air molecule.
6. TRANSFORMERS
 It provide high voltage from 220v to 50-100 kV.
7. WATER COOLING SYSTEM
 Required to prevent over heating of different part
of microscope.
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TRANSMISSION ELECTRON MICROSCOPY
WORKING
 The electron gun generate electron beam , thin tungsten filament.
 Electrons are in the form of collimated beam passes to the condenser coil & fall
on the object.
 They get scattered & transmitted to the object & pass through the objective coil
which magnifies the image of the object.
 The projector coil further magnify the image & thus final image is formed on the
fluorescence screen.
 Dense region in the specimen scatter is more and therefore appear darken in the
image where as in contrast, electron transparent regions are brighter.
MAGNIFICATION
 1,60,000x - 10,00,000x
APPLICATION
 It provides sufficient magnification and resolution to view viruses and the
internal structures of all organisms.
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29. MICROSCOPY
SCANNING ELECTRON MICROSCOPY
 This microscopes gives a typical three-
dimensional appearance.
 The illuminating system of SEM is similar
to transmission electron microscopy.
 PRINCIPLE
 It differs from TEM introducing an image
from electron emitted by object surface
rather than from transmitted electron
microscopy.
 It consist of an electron gun which
produces a finely focus beam of electron
called the primary electron beam.
 This electron passes through
electromagnetic lens & rapidly scan the
surface of specimen.
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30. MICROSCOPY
B. SCANNING ELECTRON MICROSCOPY
 When the beam of electron strikes the specimen secondary electrons are released
and transmitted to the electron collector.
 Secondary electrons are collected and use to generate a signal that produces an
image on cathode screen.
 It has a resolution of about 50Ȧ.
scanned lamp on the screen
magnification can be given by = ------------------------------------------
scanned lamp on the specimen surface
APPLICATION
 The scanning electron microscope has a wide scope in biology for
the study of small specimens, surface scanning of the cells, tissues
and membrane.
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 In medical microbiology for detecting pathogenic bacteria.
 For the detection of various types of products of microorganisms.
 It helps in examining the movement of motile cells.
 Shows greater differentiation of internal structure and clearly shows
the pellicle.
 By the use of electron microscopy structures smaller than 0.2
micrometer can be resolved.
 By the use of fluorescence microscopy rapidly detection and
identification of microbes can be done.

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 The microorganisms are so small that their study requires appropriate
methods for observing and culturing them.
 Microscopy is the technology of making very small things visible to
the human eye.
 Therefore, microscope is a major tool of the microbiologist &
biotechnologist
 Historically, it was the microscope that first revealed the secrets of
microbial structure, even today, it remains a powerful tool in
microbiological studies.
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BOOK AUTHORS YEAR EDITION
THE BIOLOGY OF
MICROORGANISM
S
2010 10th
A TEXT BOOK OF
BIOTECNOLOGY
R.C DUBEY 2008
A TEXT BOOK OF
MICROBIOLOGY
R.C DUBEY &
MAHESHWARI
CLASS NOTES
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34. THANK YOU
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