food quality control

1. Lecture 2

2. IMPORTANT TERMS
2
• Refraction
• If the light enters the glass at any angle other
than right angle, a deviation in the direction
will occur in addition to retardation, known as
Refraction.
• A curved lens will exhibit both refraction and
retardation.
• The extent of which is determined by angle of
incidence, refractive index and curvature of
the lens.

3. IMPORTANT TERMS
• Refraction
3

4. IMPORTANT TERMS
4
• Diffraction is the change in the direction and the
intensities of group of waves after passing by an
obstacle or through an aperture, whose size is
approximately the same as the wavelength of the
wave.
• Dispersion is a phenomenon, in which separation of
light into its constituent wavelength occurs from
entering a transparent medium. For example, white
light consist consists of more than one wavelength.
They would be separated or they would distribute
when they pass through prism or certain other
medium.

5. IMPORTANT TERMS
5
• Magnification is the process of enlarging an object
only in appearance and not in physical size
• Interference is the variation of wave amplitude
that occurs when waves of the same or different
frequency come together. There could be
constructive interference or there could be
destructive interference.
• Resolving Power is defined as the distance
separating 2 point objects within the specimen that
can just be distinguished from one another in the
image

6. NUMERICAL APERTURE
• The angle made by
optical axis and the
outermost rays still
covered by the
objective is the
measure of the
aperture of the
objective. It is the
half aperture angle.
6

7. NUMERICAL APERTURE
7
• Magnitude of this angle is expressed as a sine value.
The sine value of the half aperture angle multiplied
by the refractive index (η) of the medium filling the
space between the front lens and the cover slip
gives numerical aperture or NA.
• Therefore,
NA = n Sinθ
• The refractive index of the air is 1 while the
refractive index of oil immersion is 1.56.
• Hence, maximum NA of dry objective is
less than 1.0 and for oil immersion it is slightly more
than 1.0 i.e. 1.2 to 1.4.

8. LIMIT OF RESOLUTION
•
• It is the smallest distance by which two objects can
be separated and still be distinguishable as two
separate objects.
It is given by formula
where, d = limit of resolution and
• λ = wavelength of light
• Which is, 0.40 μm per blue light (min in visible range)
• 0.70 μm per red light (max in visible range)
• 0.55 μm per green light
8

9. WORKING DISTANCE:
• Working distance may be
defined as the distance
between the front lens facing
object of the objective and
the object on the slide. While
increasing magnification
working distance decreases.
Commonly it is for,
•
•
•
10x = 9mm
45x = 0.3mm
100x = 0.1mm.
9

10. TYPES OF MICROSCOPY
10
• 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

11. BRIGHTFIELD MICROSCOPY
11
• Light as source of illumination
• Glass lenses
• Limited resolution
(loses resolving power at magnifications above 2000X)
• 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 ocular
lens or eye piece located next to the eye.

12. BRIGHTFIELD 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.
Ray Diagram of Brightfield
Microscopy
12

13. DARKFIELD MICROSCOPY
• 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. Ray Diagram of Brightfield
13
icroscopy

14. DARKFIELD MICROSCOPY
• In diagnostic of microorganisms.
• 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 this 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
Ray Diagram of Brightfield
Microscopy
14

15. PHASE CONTRAST MICROSCOPY
15
• Little difference in contrast between cells and water.
Provides very high contrast as compared to the bright-field
and dark-field microscopic methods
• 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.

16. PHASE CONTRAST MICROSCOPY
• Instrumentation
• The phase-contrast microscope
consists of special condensers and
special objectives. This special
optical system makes it possible
to distinguish unstained structures
within a cell, which differs only
slightly in their refractive indices
or thicknesses.
• Applications
• Useful for microbial mobility,
shape of living cell, detection of
endospores and inclusion bodies.
16

17. FLUORESCENCE MICROSCOPY
17
• A fluorescence microscope is an optical microscope
that uses fluorescence and phosphorescence instead of,
or in addition to, reflection and absorption to study
properties of organic or inorganic substances.
• Fluorescence is the emission of light by a substance that
has absorbed light or other electromagnetic radiation
while phosphorescence is a specific type of
photoluminescence related to fluorescence.
• Unlike fluorescence, a phosphorescent material does not
immediately re-emit the radiation it absorbs.
• The fluorescence microscope was devised in the early part
of the twentieth century by August Köhler, Carl Reichert,
and Heinrich Lehmann, among others.

18. FLUORESCENCE MICROSCOPY
18

19. FLUORESCENCE MICROSCOPY
19

20. FLUORESCENCE MICROSCOPY
20

21. 21

22. ELECTRON MICROSCOPY
22

23. ELECTRON MICROSCOPY
23
Microscopy that uses a beam of accelerated electrons
as a source of illumination.
The wavelength of an electron can be up to 100,000
times shorter than that of visible light photons.
Electron microscopes have a higher resolving power
than light microscopes and can reveal the structure of
smaller objects.

24. ADVANTAGES OF ELECTRON
MICROSCOPY
24
• Advantages Of Electron Microscopy
• To study objects of >0.2 micrometer.
• For analysis of sub cellular structure.
• For study of intracellular pathogens & viruses.
• For study of cell metabolism
• For study of minute structure in nature

25. TYPES OF ELECTRON MICROSCOPE
25
There are mainly 2 types of Electron Microscope
1. Transmission Electron Microscope (TEM)
Forms images using electrons that are transmitted
through a specimen
2. Scanning Electron Microscope (SEM)
Forms image by utilizing electrons that have
bounced off the surface of the specimen

26. TRANSMISSION ELECTRON
MICROSCOPY (TEM)
26
• 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.

27. TRANSMISSION ELECTRON
MICROSCOPY (TEM)
27
• MAGNIFICATION
1,60,000x - 10,00,000x
• APPLICATION
It provides sufficient magnification and resolution to
view viruses and the internal structures of all
organisms.

28. TRANSMISSION ELECTRON
MICROSCOPY (TEM)
28
• 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.

29. TRANSMISSION ELECTRON
MICROSCOPY (TEM)
29

30. SCANNING ELECTRON MICROSCOPY
(TEM)
30
• 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.

31. SCANNING ELECTRON MICROSCOPY
(TEM)
31
• 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.

32. SCANNING ELECTRON MICROSCOPY
(TEM)
32

33. SCANNING ELECTRON MICROSCOPY
(TEM)
33
• 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.
• In medical microbiology for detecting pathogenic
bacteria.
• For the detection of various types of products of
microorganisms.
• 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.

34. DIFFERENCES BETWEEN SEM AND TEM
34

35. DIFFERENCES BETWEEN OPTICAL (LIGHT)
MICROSCOPY AND ELECTRON MCROSCOPY
35

36. 36