department of biochemistry (pg) bioanalytical techniques ...pg)-10.p… · using a confocal...
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2D Gel Electrophoresis
KONGUNADU ARTS AND SCIENCE COLLEGE
(AUTONOMOUS)
Coimbatore – 641 029
Bioanalytical Techniques
Dr. S. Viswanathan
U/TS/649
DEPARTMENT OF BIOCHEMISTRY (PG)
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• INTRODUCTION
• DEFINITION
• HISTORICAL BACKGROUND
• VARIBLES USED IN MICROSCOPY
• COMPOUND MICROSCOPE - Structure and
Function
• USE OF MICROSCOPE
• VARIOUS TYPES OF MICROSCOPES
• CARE OF MICROSCOPE
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INTRODUCTIONTO
MICROSCOPY3
• Understanding the
optical principles
and construction of
microscopes
• Role of microscopy
• Microscopic
techniques and
application
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DEFINITION 4
• A microscope (Greek: micron = small and
scopos = aim)
• MICROSCOPE - An instrument for viewing
objects that are too small to be seen by the
naked or unaided eye
• MICROSCOPY - The science of
investigating small objects using such an
instrument is called microscopy
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HISTORICAL BACKGROUND5
• 1590 - Hans Janssen and his son Zacharias
Janssen, developed first microscope.
• 1609 - Galileo Galilei - occhiolino or compound
microscope.
• 1620 - Christian Huygens, another Dutchman,
developed a simple 2-lens ocular system that
was chromatically corrected.
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Anton van Leeuwenhoek
(1632-1723)6
• Anton van Leeuwenhoek is
generally credited with
bringing the microscope to
the attention of biologists.
• A tradesman of Delft, Holland.
1661 - He discovered bacteria, free-living
and parasitic microscopic protists, sperm
cells, blood cells, microscopic nematodes
etc.
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Microscope used by
Anton von Leeuwenhoek7
An old pocket Microscope
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9
VARIABLES
USEDIN
MICROSCOP
Y
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MAGNIFICATION 9
• Degree of enlargement
• No of times the length, breadth or
diameter, of an object is multiplied.
MAGNIFICATION VS SHARPNESS
• USEFUL MAGNIFICATION AND EMPTY
MAGNIFICATION
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• RESOLUTION – Ability to reveal closely
adjacent structural details as separate and
distinct
• LIMIT OF RESOLUTION (LR) – The min
distance between two visible bodies at
which they can be seen as separate and
not in contact with each other
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Types of microscope Resolving power
Compound Microscope 200 nanometers
Scanning Electron
Microscope
10 nanometers
Transmission Electron
Microscope
0.2 nanometers
W = Wavelength
NA = Num aperture11
• LR = 0.61 x WNA
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NUMERICAL APERTURE(NA)
• Ratio of diameter of lens to its focal length
• NA = n Sin θ/2n = refractive index,
θ/2
A
B
D
θ = angle of aperture (CAD)
C
• n of air = 1
• n of oil = 1.5
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DEFINITION
• Capacity of an objective to render outline
of the image of an object clear and distinct
• Depends of elimination of Spherical and
Chromatic aberration
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ABERRATION
• Chromatic aberration
• Correction of aberration – Achromatic
objective and Apochromatic objectives.
Blue focusRed focus
Incident
light
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• Spherical aberration
Focus of marginal rays
Focus of axial rays
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TYPES OF MICROSCOPE16
• Simple microscope
• Compound microscope
• Phase Contrast
Microscope
• Dark Ground Microscope
• Fluorescent Microscope
• Electron Microscope
• Others
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COMPOUND
MICROSCOPE
17
Compound microscope made
by John Cuff 1750
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PARTS OF COMPOUND MICROSCOPE
• Ocular (Eye piece)
• Body or Tube
• Coarse focusing knob
• Fine focusing knob
• Objective Lens
• Movable stage
• Condenser Lenses
• Field (Iris) Diaphragm
• Mirror and light source
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OBJECTIVE LENS
• Mounted on Nose piece
• It forms magnified real image.
• Magnification of objective
= Optical Tube length
Focal Length
•
•
•
•
Scan - 4X
Low Power - 10X
High Power - 40X
Oil immersion - 100
TYPES
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OIL IMMERSIONOBJECTIVE
Highest magnification
Oil prevents refraction of light outwards and
allows it to pass straight in to objective G
GLASS
OIL
AB
C
E
F
FBEG - OILD
ABCD - AIR
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EYE PIECE21
•
•
Forms magnified virtual & erect image
TYPES
(a)
(b)
(c)
(a)
(b)
(c)
Monocular
Binocular
Trinocular
or
Huygenian
Ramsden
Compensating
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PARTS OF MICROSCOPE (Cont)
Iris Diaphragm
Condenser
Field Diaphragm
Diaphragm control ring
Focusing knob
illumination source
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ILLUMINATION - Lamp,
sunlight, battery operated
lamp, 60 W bulb, Quartz
halogen light.
FILTERS - Blue, Green,
Heat absorbing filters,
Barrier filters.
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Multiple step operation employed to attain optimal
illumination:
1. Remove any diffusing filter.
2. Put a slide on the stage and focus.
3. Completely close the field diaphragm .
4.Move the condenser until the border or the iris
hexagon is neat and clear.5. Center if necessary.
6.Open the field diaphragm until the tip of the
hexagon touches the field limit
KOHLER’S ILLUMINATION 25
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HOW A MICROSCOPE WORKS
?
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OPTICAL PATH IN COMPOUND
MICROSCOPE
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METHODOF USING
COMPOUND
MICROSCOPE
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1. Grasp the microscopes arm with
one hand and place your other
hand under the base.
29
2. Place the microscope on a bench.
Adjust seat
3. Clean Lenses.
4. Turn the coarse adjustment knob
to raise the body tube
arm
arm
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5. Revolve the nose piece to set low-power objective lens.
6. Adjust the Condenser lenses and diaphragm .
7. Place a slide on the stage and secure with stage clips.
8. Switch on the light at low intensity and then increase intensity.
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9. Turn the coarse adjustment knob to lower the body tube until the low power objective reaches its lowest point.
10.Looking through the eyepiece, very slowly move
the coarse adjustment knob until the specimen
comes into focus.
11. Adjust distance between eye piece.32
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12. Switch to the high power objective lens only
after adjusting condenser and iris diaphragm.
13. Place a drop of oil over specimen before using
oil immersion objective.
14. Lower the objective until oil makes contact with
objective.
15. Looking through the eyepiece, very slowly
focus the objective away from the slide i.e by
raising the objective lens.
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HOW TO OBSERVE A SLIDE ?33
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CAUSES OF ERROR IN
FOCUSING34
• Revolving Nose Piece is off centre
• Preparation is upside down
• Thick cover slip
• Dirt or Dried oil over Lens
• Air bubble in immersion oil
• Poor illumination – Condenser not fully
racked up
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PHASE CONTRAST MICROSCOPE35
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PHASE CONTRAST
MICROSCOPE36
• First described in 1934 by Dutch physicist
Frits Zernike
• Produces high-contrast images of
transparent specimens
• Advantage - Living cells can be examined
in their natural state
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PRINCIPLE OF PHASE CONTRAST
MICROSCOPY37
• Unstained bacteria have constituents of
different refractive index .
• Diffraction of light
• Phase contrast microscope employs an
optical mechanism to translate minute
variations in phase into corresponding
changes in intensity of image.
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REQUISITE FOR PHASE
CONTRAST MICROSCOPY
39
• ANNULAR DIAPHRAGM
• PHASE PLATE
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CONDENSER ANNULUS 40
• The condenser annulus or annular
diaphragm is opaque flat-black (light
absorbing) plate with a transparent
annular ring.
• Produces hollow cone of light.
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Condenser
annulus
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PHASE PLATE 42
• Placed in back focal plane of objective.
• FUNCTION :-
1. Enhances phase difference by
retarding diffracted wave front by one
quarter of wavelength .
2. Reduces intensity of direct rays and
equalizes it with diffracted rays intensity.
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Phase plate
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IMAGES OF PHASE CONTRAST
MICROSCOPY
Clostridium botulinum45Spirilium volutans
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COMPARISION OF IMAGES OF BRIGHT
FIELD AND PHASE CONTRAST
MICROSCOPY
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USES OF PHASE CONTRAST
MICROSCOPY46
• Phase contrast enables visualization of
internal cellular components.
• Diagnosis of tumor cells .
• Examination of growth, dynamics, and
behavior of a wide variety of living cells in
cell culture
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DARK GROUND MICROSCOPE 47
• Optical system to enhance the contrast of
unstained bodies .
• Specimen appears gleaming bright
against dark background
• PRINCIPLE OF DGI
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OPTICAL PATH IN DARK GROUND
MICROSCOPY 48
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REQUISITES FOR DARK GROUND
MICROSCOPY
• Dark ground
condenser
• High intensity
lamp
• Funnel stop
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USES OF DARK GROUND
MICROSCOPY50
• Useful in demonstrating
-Treponema pallidum
- Leptospira
- Campylobacter jejuni
- Endospore
Treponema pallidum
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FLUORESCENCE MICROSCOPY51
• PRINCIPLE
UV light
Fluorochrome
Visible
radiation
FITC EX - 495 nm EM - 520nm
TRITC EX – 540 nm EM – 590 nm
Texas Red Ex – 600 nm EM – 615 nm
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• UV rays passes through exciter filter
• Dark ground condenser
• Micro organisms stained with fluorescent
dye, when examined under microscope
with ultraviolet light are seen as bright
object against dark background
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USE OF FLUORESCENCE
MICROSCOPY53
• Auramine Rhodamine –Yellow Fl - Tubercle bacilli
• Acridine Orange R - gives
orange red Fl with RNA and
yellow green Fl with DNA
• QBC
• IMMUNOFLUORESCENCE
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ELECTRON
MICROSCOPE
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ELECTRON
MICROSCOP
E
55
• Electron Microscopes uses a beam of highly
energetic electrons to examine objects on a
very fine scale. This examination can yield
the info about
– Topography
– Morphology
– Composition
– Crystallographic structure
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TYPES OF EM 56
• Transmission Electron Microscope (TEM)
• Scanning Electron Microscope (SEM)
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TRANSMISSION
ELECTRON
MICROSCOPE (TEM)• Stream of electrons is formed
• Accelerated using a positive electrical potential
• Focused by metallic aperture and Electro magnets
• Interactions occur inside the irradiated sample which are detected and transformed into an image .
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TEM
(Cont)
58
• Projector Lens forms
image on Fluorescent
viewing screen
• 2D Image
• Magnification
10,000 X to 100,000 X
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• Scan a gold-plated specimen to give a 3-D
view of the surface of an object which is
black and white.
• Used to study surface features of cells and
viruses.
• Scanning Electron microscope has
resolution 1000 times better than Light
microscope .
SCANNING ELECTRON
MICROSCOPE
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WORKING OF SEM 60
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SEM IMAGES
Vibrio cholerae with
polar flagellaTreponema pallidum
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INVERTED MICROSCOPE
• Used in metallurgy
• Examination of cultures in flat bottom
dishes
• Micro dissection
• Examination of parasites
• Observation of agglutination in serology
OTHER MICROSCOPES
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STEREO MICROSCOPE
• Double Microscope
• Produces 3D images
POLARIZING MICROSCOPE
• Uses two Polariser
• Gives info about Birefringence of a body
• Used in Crystallography, Urine examination
• Apple Green Birefringerence in AMYLODOS64IS
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• Uses a laser beam to illuminate
a specimen whose image is
then digitally enhanced for
viewing on a computer monitor.
• Laser beam scans single plane
of 1µm thickness.
CONFOCAL SCANNING
LASER MICROSCOPE64
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Comparison of Depth of
Light Collection and
Image clarity
65
Light Microscope Confocal Scanning
Laser Microscope
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PRINCIPLE OF CONFOCAL MICROSCOPY66
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USES OF CONFOCAL
MICROSCOPE67
• Observing cellular morphology in
multilayered specimen
• Eg. used in diagnosing Ca cervix
• Evaluation and diagnosis of basal cell
carcinoma of skin
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What is the advantage of
using a confocal
microscope?
68
• By having a confocal pinhole, the
microscope is really efficient at rejecting
out of focus fluorescent light so that very
thin section of a sample can be analyzed.
• By scanning many thin sections through a
sample, one can build up a very clean
three-dimensional image .
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NEWER MICROSCOPE 69
• SCANNING PROBE MICROSCOPE -Class of
Microscope that measures surface features
by moving a sharp probe over object surface.
Used to visualize atoms and molecules
– Scanning Tunneling Microscope (STM)
– Atomic Force Microscope (AFM)
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CARE OF THE
MICROSCOPE
70
• Handling
• Proper storage
• Care of Lenses
• Care of oil emersion objective
• Care of lamp