A microscope (from the Ancient Greek: micro- "small“ and scope-"to look") is an instrument used to see objects that are too small for the naked eye

1. Principles of Microscopy
Dr Smitha Vijayan
Associate Professor, School of Biosciences
Mar Athanasios College For Advanced Studies Tiruvalla (MACFAST)

2. What IsA Microscope ?
•A microscope (from the Ancient Greek: micro- "small“ and scope-"to
look") is an instrument used to see objects that are too small for the
naked eye.
•A microscope is an instrument that produces an accurately enlarged
image of small objects.
•The science of investigating small objects using such an instrument is
called
microscopy.
•Microscopic means invisible to the eye unless aided by a microscope.

3. ◉ Our eyes cannot focus on objects nearer than about 25 cm (i.e., about 10 inches).
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.
◉ Lens strength is related to focal length; a lens with a short focal length magnifies
an object more than a lens having a longer focal length.
Why Need
Microscope!
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4. Light microscopes: first microscopes invented, most commonly
used type.
To understand light microscopy, we must consider the way lenses
bend and focus
light to form images.
When a ray of light passes from one medium to another, refraction
occurs; that is, the ray is bent at the interface.
The refractive index is a measure of how much a substance slows
the velocity of light; the direction and magnitude of bending are
determined by the refractive indices of the two media forming the
interface.
For example, when light passes from air into glass, which has a
greater refractive index, it is slowed and bent toward the normal, a
line perpendicular to the surface
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5. There Are Several Types
of Light Microscopes
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Bright-Field Microscope: Dark
Object, Bright Background:
used to examine both
stained and unstained
specimens. It forms a dark
image against
a lighter background, thus it
has a “bright field.” It consists
of a
metal stand composed of a
base and an arm to which the
remaining
parts are attached

6. A light source: a mirroror an electric illuminator is located in the base.
Two focusing knobs: the fine and coarse adjustment knobs, are located
on the arm
The stage: positioned about halfway up the arm, Microscope slides are
clipped to the stage, which can be moved during viewing by rotating
control knobs.
The substage condenser lens (or simply, condenser) is within or beneath
the stage and
focuses a cone of light on the slide. Its position may be fixed in simpler
microscopes but can be adjusted vertically in more advanced models.
The curved upper part of the arm holds the body assembly, to which a
nosepiece and one or more ocular lenses (also called eyepieces) are
attached.
Binocular microscopes have eyepieces for both eyes.
The body assembly contains a series of mirrors and prisms so the barrel
holding the eyepiece may be tilted for ease in viewing.
The nosepiece holds three to five objective lenses of differing
magnifying power and can be rotated to change magnification 6

7. parfocal
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A microscope should be parfocal:that is, the image should remain in
focus when objective lenses are changed.
The image seen when viewing a specimen with a compound
microscope is created by the objective and ocular lenses working
together. Light from the illuminated specimen is focused by
the objective lens, creating an enlarged image within the microscope.
The ocular lens further magnifies this primary image.
The total magnification is calculated by multiplying the
objective and eyepiece magnifications together.
For example, if a 45× objective lens is used with a 10× eyepiece, the
overall magnification of the specimen is 450×.

8. “
◉ Better Microscope Resolution Means a Clearer Image
◉ The most important part of the microscope is the objective lens,which must
produce a clear image, not just a magnified one.
◉ Resolution is the ability of a lens to separate or distinguish between small
objects that are close together.
◉ At best, the resolution of a bright-field microscope is 0.2 μm, which is about
the size of a very small bacterium.
◉ Resolution is in part dependent on the numerical aperture (n sin θ) of a
lens.
◉ Numerical aperture is defined by two components: n is the refractive index
of the medium in which the lens works (e.g., air = 1) and θ is 1/2 the angle
of the cone of light entering an objective
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To increase the refractive index : immersion oil, a colorless
liquid with the same refractive index as glass
If air is replaced with immersion oil, many light rays that would
otherwise not enter the objective due to reflection and refraction at
the surfaces of the objective lens and slide will now do so
Bright-field microscopes are probably the most common
microscope found in teaching, research, and clinical
laboratories.
Three types of light microscopes create detailed, clear
images of living specimens:
dark-field microscopes, phase-contrast microscopes, and
differential interference contrast microscopes.
In dark-field microscopy, a dark-field stop (inset) is placed
underneath the condenser lens system.
The condenser then produces a hollow cone of light so that the only
light entering the
objective is reflected or refracted by the specimen.

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The dark-field microscope produces detailed images
of living, unstained cells and organisms by simply
changing the way in which they are illuminated

11. ◉ The refractive indices of bacterial
cell structures are greater than
that of water.
◉ Light waves passing through a
cell structure will be diffracted and
slowed more than light waves
passing through the water inside
and outside
Phase-Contrast Microscope
◉ both deviated light waves that interact with
bacterial cell structures and undeviated
light waves that pass around and through
the cell are produced.
◉ Because the deviated light waves are
slowed relative to the undeviated light
waves, they are said to be out of phase.
◉ That is, the crests and troughs of the
deviated and undeviated waves do not
align.
◉ Typically the deviated light waves are
slowed by about ¼ wavelength compared
to the undeviated light 11

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Phase-contrast microscopes take advantage
of this phenomenon to create differences
in light intensity that provide contrast to allow
the viewer to see a clearer, more detailed image
of the specimen. A condenser annulus and a phase plate

13. ◉ The condenser annulus is an opaque disk with a thin transparent ring.
◉ A ring of light is directed by the condenser annulus to the condenser, which focuses
the light on the specimen
◉ Deviated and undeviated light then pass through the objective toward the phase
plate.
◉ The phase plate has a thin ring through which the undeviated light (i.e., from the
surroundings) is focused
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Differential Interference Contrast Microscope
The differential interference contrast (DIC) microscope is
similar to the phase-contrast microscope in that it creates an image
by detecting differences in refractive indices and thickness.
Two beams of plane-polarized light at right angles to each other
are generated by prisms.
In one design, the object beam passes
through the specimen, while the reference beam passes through
a clear area of the slide. After passing through the specimen, the
two beams combine and interfere with each other to form
an image. A live, unstained specimen appears brightly colored
and seems to pop out from the background, giving the viewer
the sense that a three-dimensional image is being viewed
Structures such as cell walls, endospores, granules,
vacuoles, and nuclei are clearly visible.

16. Fluorescence
Microscopes
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Use Emitted Light to Create Images
An object also can be seen because it emits light.
When some molecules absorb radiant energy, they become excited and
release much of their trapped energy as light. Any light emitted by an excited
molecule has a longer wavelength (i.e., has lower energy) than the radiation
originally absorbed.
Fluorescent light is emitted very quickly by the excited molecule as it gives
up its trapped energy and returns to a more stable state.
The fluorescence microscope excites a specimen with a specific wavelength
of light that triggers the emission of fluorescent light by the object, which forms
the image
Specimens are stained with fluorochromes
The fluorochrome absorbs light energy from the excitation light and emits
fluorescent light that travels up through the objective lens into the microscope

17. To visualize photosynthetic microbes, as their pigments
naturally fluoresce when excited by light of specific
wavelengths. It is even possible to distinguish live bacteria
from dead bacteria by the color they fluoresce after treatment
with a specific mixture of stains
Another important use of fluorescence microscopy is the
localization of specific proteins within cells.
Confocal Microscopy
The confocal microscope uses a laser beam to illuminate a specimen that has been
fluorescently stained. A major component of the confocal microscope is an opening (that is, an
aperture) placed above the objective lens.
The aperture eliminates stray light from parts of the specimen that lie above and below the
plane of focus Thus the only light used to create the image is from the plane of focus, and a
much sharper image is formed. To generate a confocal image, a computer interfaced with the
confocal microscope receives digitized information from each plane in the specimen. This
information can be used to
create a composite image that is very clear and detailed

18. ◉ Microscopes that use electrons as the light source and electromagnetic
coils to direct the path of the e- are called as electron microscopes.
◉ (The optical system is completely replaced by electromagnetic
coils).
◉ The first electron microscope was designed by Knoll and Ruska (1931).
◉ (Wavelength of e- = 0.05A very short wavelength with very high
◉ magnification).
◉ The magnification of electron microscope is 1000 times higher than the
◉ light microscope. (Therefore the magnification of e- is 100 × 1000 =
1,00,000 X.)
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Electron Microscope

19. Types of Electron Microscope
Transmission electron microscope ( TEM )
Scanning electron microscope ( SEM)
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Transmission electron microscope (TEM)
In this type e- are allowed to transmit through the specimen is called TEM.
The first TEM was designed by Max Knoll and Ernst Ruska (1931).

20. Basic principle
◉ Similar to the compound microscopes but the e- beam is substituted for
light source and electromagnetic coils to optical lens.
◉ When high voltage current is passed through the cathode ray tube, e-
beams are produced.
◉ Electromagnetic coils direct the e- beams to pass through the specimen.
◉ It is stained with gold or osmium and the image is collected by objective
lens and amplifier (electromagnetic coils).
◉ The image cannot be seen by our naked eye, so it is casted on a screen or
photographic plate or camera.
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Electron microscopes are kept in
vacuum because
1. Electrons are easily absorbed in air.
2. Electrons are move in a straight line only in vacuum.
Instrumentation: The TEM has an electron gun and condenser lens.
A) Electron gun
B) Condenser lens
C) Objective lens
D) Amplifier lens
E) Projector lens
F) Ancillary equipment

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23. Electron gun:
◉ Made up of cathode ray tube with tungsten filament (2mm long)
◉ Located at the top of the microscope.
◉ It generates e
Condenser lens
◉ Two condenser lens or electromagnetic coils are present below the e
gun.
◉ They collect and direct the beams into the specimens on a stage.
◉ A thin section of specimen is placed on a thin plastic film mounted on
a copper gird (3 mm diameter).
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24. Objective lens
◉ It is an electromagnetic coil placed below the specimen stage.
◉ It collects the specimen image and focus towards the amplifier lens.
Amplifier lens
◉ It is an electromagnetic coil below the objective lens and magnifies the image several times
◉ Projector lens
◉ collects the magnified image and focused on a fluorescent screen or photographic plate
Ancillary equipment
◉ The entire set up is placed in a vacuum tube.
◉ TEM release large amount of heat during working hours, so cooling system is present
◉ It needs high power supply.
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25. Preparation of specimen for
TEM:
◉ Biological material contains low atomic weight elements like
carbon,hydrogen, oxygen and nitrogen.
◉ They do not give high resolution.
◉ Therefore, the biological sample has to be loaded with heavy
atoms like gold or osmium and these atoms protect the
specimen from destruction.
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26. ◉ Applications
◉ TEM is an ideal tool for the study of ultra structure of a cell.
◉ It is used to identify plant and animal virus.
◉ It is widely applied in various researches in oncology, pollution,
biochemistry, molecular biology, etc.
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27. Disadvantages:
◉ Very high cost.
◉ We cannot study 3 dimensional structures of the
specimens.
◉ The specimens should be fixed properly and should take
ultra thin sections, because an electron has limited
penetrating power.
◉ We could not study live specimens
◉ It is successful only under high vacuum condition.
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28. Scanning Electron
Microscope
In SEM, the surface of the specimen is scanned by electron beam.
This was first designed by Max Knoll (1935).

29. Principle:
◉ SEM use electron beam for illumination and electromagnetic coils for directing
the path of e- beam.
◉ When e- is focused on the specimen, it produces secondary e- (SE), back
scattered e- (BSE) and characteristic X-rays.
◉ Secondary electrons are reflected due to the interactions between atoms in
specimens and e- beam.
◉ Back scattered e- gives information about the distribution of different
elements.
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31. Instrumentation:
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Electron gun:
it is the source of e- beam and located at the top of the microscope.
It consists of cathode plate and anode plate.
Condenser lens:
There are two condenser lenses just below the e- gun.
They collect and concentrate the e- in to a strong beam
Deflection coils:
below the condensers, there is a deflection coil to direct the beam of e- in
to the specimen stage
Specimen stage:
it is present in slanting position at the lower side of deflection coil
Separate e- detectors
( scintillator& PMT ) are attached in the vacuum tube.
Electronic amplifiers are connected with detectors.
The electric signals are converted into bright spots of varying density by scanning circuit.

32. Additional things
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Image is displayed on a photographic plate or computer monitor.
The entire set up should be placed in a vacuum tube.
Power supply with high voltage.
SEM releases huge amount of heat, so cooling system is present around it.
Dry materials like wood, bone, feathers, insect’s wings
and shells are coated with thin film of electro
conductive materials like gold, platinum, tungsten,
osmium, chromium and graphite. Then the specimens
are placed on the stage.

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Advantages:
SEM is use full to view the surface of
microorganisms (Bacteria, Diatoms), pollen
grains, hairs and scales of plants and animals.
It is free of chromatic aberrations.
It produce 3D image.
SEM is used study archeological specimens
and
fossils.
It is used to analyze the compound eyes of
insects.
Disadvantages:
Lower resolution than TEM.
High cost.
Complete vacuum is needed.
Factors limit the quality is
uncontrolled emission of e- and
scan faults.

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