2. - Introduction
- Definition
- Historical Background
- Varibles Used In Microscopy
- Compound Microscope - Structure And Function
- Use Of Microscope
- Various Types Of Microscopes
- Care Of Microscope
2
3. INTRODUCTION TO
MICROSCOPY
- Understanding the
optical principles and
construction of
microscopes
- Role of microscopy
- Microscopic
techniques and
application 3
4. DEFINITION
- A microscope (Greek: micron = small and scopos =
to look)
- MICROSCOPE: Is 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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5. HISTORICAL BACKGROUND
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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6. Antony van Leeuwenhoek
(1632-1723)
- 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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9. MAGNIFICATION
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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10. 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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11. LR = 0.61 x W W = Wavelength
NA NA = Num aperture
Types of microscope Resolving power
Compound Microscope 200 nanometers
Scanning Electron Microscope 10 nanometers
Transmission Electron Microscope 0.2 nanometers
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12. NUMERICAL APERTURE(NA)
- Ratio of diameter of lens to its focal length
- NA = n Sin θ/2
n = refractive index,
θ = angle of aperture (CAD) B
C D
θ/2
n of air = 1
n of oil = 1.5
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A
13. 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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14. ABERRATION
- Chromatic aberration
- Correction of aberration – Achromatic
objective and Apochromatic objectives.
Incident
light
Blue focus
Red focus 14
18. 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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19. OBJECTIVE LENS
• Mounted on Nose piece
It forms magnified real image.
• Magnification of objective
= Optical Tube length
Focal Length
• Scan - 4X
• Low Power - 10X
TYPES
• High Power - 40X
• Oil immersion - 100X 19
20. OIL IMMERSION OBJECTIVE
- Highest magnification
- Oil prevents refraction of light outwards and
allows it to pass straight in to objective
G
FBEG - OIL D
ABCD - AIR
E
OIL
C GLASS
B 20
A
22. What’s the power of this lens?
To calculate the power of magnification, multiply the power of the
ocular lens by the power of the objective, e..g.: 10x40=400 times
What are the powers of
magnification for each
of
the objectives we have
on our microscopes?
Fill in the table on
your worksheet.
24. Comparing Powers of Magnification
We can see better details with higher the powers of
magnification, but we cannot see as much of the image.
10x 40x
Which of these images
would be viewed at a
higher power of
magnification?
25. Let’s give it a try ...
1 – Turn on the microscope and then rotate the nosepiece to click the
red-banded objective into place.
2 – Place a slide on the stage and secure it using the stage clips. Use
the coarse adjustment knob (large knob) to get it the image into view
and then use the fine adjustment knob (small knob) to make it
clearer.
3 – Once you have the image in view, rotate the nosepiece to view it
under different powers. Draw what you see on your worksheet!
Be careful with the largest objective! Sometimes there is
not enough room and you will not be able to use it!
4 – When you are done, turn off the microscope and put up the
slides you used.
26. How to make a wet-mount slide …
1 – Get a clean slide and coverslip from your teacher.
2 – Place ONE drop of water in the middle of the slide. Don’t use
too much or the water will run off the edge and make a mess!
3 – Place the edge of the cover slip on one side of the water drop.
4 - Slowly lower the cover slip on top of the drop.
Cover Lower slowly
Slip
5 – Place the slide on the stage and view it first with the red-banded
objective. Once you see the image, you can rotate the nosepiece to
view the slide with the different objectives.
You do not need to use the stage clips
when viewing wet-mount slides!
28. KOHLER’S ILLUMINATION
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
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30. The Parts of a Microscope
Ocular Lens
Body Tube
Nose Piece
Arm
Objective
Lenses
Stage
Stage
Clips Coarse Adj
Diaphragm Fine Adjustment
Light Source
Base
31. Body Tube
The body tube holds the objective lenses
and the ocular lens at the proper distance
Diagram
32. Nose Piece
The Nose Piece holds the objective lenses
and can be turned to increase the
magnification
Diagram
36. Light Source
Projects light upwards through the
diaphragm, the specimen and the lenses
Some have lights, others have mirrors
where you must move the mirror to reflect
light
Diagram
44. Magnification
To determine your magnification…you
just multiply the ocular lens by the
objective lens
Ocular 10x Objective 40x:10 x 40 = 400
So the object is 400 times “larger”
Objective Lens have
their magnification
written on them.
Ocular lenses usually magnifies by 10x
48. 1. Grasp the microscopes arm with
one hand and place your other
hand under the base.
2. Place the microscope on a bench.
arm
arm Adjust seat
3. Clean Lenses.
4. Turn the coarse adjustment knob
to raise the body tube
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49. 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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50. 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.
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51. 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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53. Causes of Error in Focusing
- 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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55. Phase Contrast Microscope
- 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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56. Principle Of Phase Contrast
Microscopy
- 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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59. Condenser Annulus
- 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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61. Phase Plate
- 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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65. Uses of Phase Contrast Microscopy
- 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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66. Dark Ground Microscope
- Optical system to enhance the contrast of
unstained bodies .
- Specimen appears gleaming bright against
dark background
PRINCIPLE OF DGI
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68. Requisites for Dark Ground
Microscopy
- Dark ground
condenser
- High intensity
lamp
- Funnel stop
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69. Uses of Dark Ground Microscopy
Useful in demonstrating
-Treponema pallidum
- Leptospira
- Campylobacter jejuni
- Endospore
Treponema pallidum
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70. Fluorescence Microscopy
PRINCIPLE
Visible
UV light
radiation
Fluorochrome
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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71. - 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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72. Use of Fluorescence Microscopy
- 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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74. ELECTRON MICROSCOPE
-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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75. TYPES OF EM
- Transmission Electron Microscope (TEM)
- Scanning Electron Microscope (SEM)
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76. 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 . 76
77. TEM (Cont)
-Projector Lens forms
image on Fluorescent
viewing screen
- 2D Image
- Magnification
10,000 X to 100,000 X
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78. Scanning Electron Microscope
- 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 .
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80. SEM IMAGES
Vibrio cholerae with
Treponema pallidum polar flagella 80
81. OTHER MICROSCOPES
INVERTED MICROSCOPE
- Used in metallurgy
- Examination of cultures in flat bottom
dishes
- Micro dissection
- Examination of parasites
- Observation of agglutination in serology
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82. 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
82
AMYLODOSIS
83. CONFOCAL SCANNING
LASER MICROSCOPE
- 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.
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84. Comparison of Depth of Light
Collection and Image clarity
Light Microscope Confocal Scanning
Laser Microscope
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86. USES OF CONFOCAL
MICROSCOPE
- 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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87. What is the advantage of using a
confocal microscope?
- 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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88. NEWER MICROSCOPE
- 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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89. CARE OF THE MICROSCOPE
- Handling
- Proper storage
- Care of Lenses
- Care of oil emersion objective
- Care of lamp
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