during this ppt of microscopes we will be able to know
INTRODUCTION
DEFINITION
HISTORICAL BACKGROUND
VARIABLES USED IN MICROSCOPY
VARIOUS TYPES OF MICROSCOPES
COMPOUND MICROSCOPE - Structure and Function
USE OF MICROSCOPE
CARE OF MICROSCOPE
defintion
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
2. My work, which I've done for a long time, was not pursued in
order to gain the praise I now enjoy,
but chiefly from a craving after knowledge, which I notice
resides in me more than in most other men.
Whenever I found out anything remarkable, I have thought it
my duty to put down my discovery on paper,
so that all people might be informed thereof.
Antony van Leeuwenhoek. Letter of June 12, 1716
3. • INTRODUCTION
• DEFINITION
• HISTORICAL BACKGROUND
• VARIABLES USED IN
MICROSCOPY
• VARIOUS TYPES OF
MICROSCOPES
• COMPOUND MICROSCOPE -
Structure and Function
• USE OF MICROSCOPE
• CARE OF MICROSCOPE
MICROSCOPY
4. 4
INTRODUCTION TO MICROSCOPY
• Understanding the optical
principles and
construction of
microscopes
• Role of microscopy
• Microscopic techniques
and application
5.
6. 6
DEFINITION
• 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
7. 7
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.
8. 8
Anton 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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MAGNIFICATION
• Degree of enlargement
• No of times the length, breadth
or diameter, of an object is
multiplied.
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Types of microscope Resolving power
Compound Microscope 200 nanometers
Scanning Electron
Microscope
10 nanometers
Transmission Electron
Microscope
0.2 nanometers
RESOLUTION
Ability to reveal closely adjacent structural details
as separate and distinct
14. RESOLVING POWER
Optical Instrument Resolving
Power
R.P. in
Angstroms
Human eye 0.2 mm 2,000,000 Å
Light microscope 0.2 m 2000 Å
Scanning electron
microscope
5-10 nm 50-100 Å
Transmission
electron
microscope
0.5 nm 5 Å
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TYPES OF MICROSCOPE
• Simple microscope
• Compound microscope
• Phase Contrast
Microscope
• Dark Ground Microscope
• Fluorescent Microscope
• Electron Microscope
• Others
21. Light Path
• Light enters objective lens
• Collect light from specimen
• Forms a magnified inverted
image
• Image magnified by ocular lens
& passed to eye
• Total magnification (40x X 10x =
400x)
• Parfocal
22. Oil Immersion Lens
• Light bends (refracts) as it passes
from glass into air
• Some light does not enter this
smaller objective lens
• Use oil between slide and 100x lens
• Displaces air between lens and
specimen
• Glass and oil have same RI so
less bending
• Oil becomes part of the optics of
glass
• Increases resolution
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1. Grasp the microscopes arm with one hand
and place your other hand under the base.
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.
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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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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
32. Smear of gram positive bacteria with cocci and
bacilli shape, under 100X light microscope.
33. DARK FIELD MICROSCOPY
• special condenser diaphragm
• occludes direct light,
passes wide angle light
• angle too wide to enter
objective
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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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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
37. 37
DARK GROUND MICROSCOPE
• Optical system to enhance the contrast of unstained
bodies .
• Specimen appears gleaming bright against dark
background
PRINCIPLE OF DGI
38. 38
USES OF DARK GROUND MICROSCOPY
Treponema pallidum
Useful in demonstrating
-Treponema pallidum
- Leptospira
- Campylobacter jejuni
- Endospore
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FLUORESCENCE MICROSCOPY
• 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
40. 40
• 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
MICROSCOPY
• Auramine Rhodamine – Yellow Fl -
Tubercle bacilli
• Acridine Orange R - gives orange red Fl
with RNA and yellow green Fl with DNA
• QBC
• IMMUNOFLUORESCENCE
43. 43
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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TYPES OF EM
• Transmission Electron Microscope (TEM)
• Scanning Electron Microscope (SEM)
45. 45
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 .
46. 46
TEM (Cont)
• Projector Lens forms
image on Fluorescent
viewing screen
• 2D Image
• Magnification
10,000 X to 100,000 X
47. 47
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
50. 50
INVERTED MICROSCOPE
• Examination of cultures in flat bottom dishes
• Micro dissection
• Examination of parasites
• Observation of agglutination in serology
OTHER MICROSCOPES
51. 51
CARE OF THE MICROSCOPE
• Handling
• Proper storage
• Care of Lenses
• Care of oil emersion objective
• Care of lamp