The document discusses different types of light microscopy. It describes the basic components and workings of simple and compound light microscopes. Various techniques used in light microscopy are also summarized, including bright field, dark field, phase contrast, and differential interference contrast microscopy. Specific applications and advantages/disadvantages of each technique are highlighted. A brief history of developments in light microscopy and important optical concepts such as magnification and resolution are also provided.

1. LIGHT MICROSCOPY
Dr.Balaji P,
First year Post Graduate,
M.D., Microbiology,
Dept. of Microbiology,
Chengalpattu Medical college.
Dr.Balaji P CHMC

2. Dr.Balaji P CHMC

3. Dr.Balaji P CHMC

4. History
• 1st century AD
• Lentil
• Burning glasses /magnifying glasses 6 X – 10 X
• 1590 Dutch , son and father – lenses in tube
• Galileo –principles
• Antony von leeuwenhoek 270 X
• Bacteria, yeast, Blood cells, tiny animals in
water
Dr.Balaji P CHMC

5. OPTICAL/LIGHT MICROSCOPE
• Microscopes which uses visible light
and lens(es) to magnify small objects
• Two types :
a) Simple
b) Compound
Dr.Balaji P CHMC

6. Simple Microscope
• This is the type of microscope which was
invented first
• Original design of Light microscope
• Uses single lens
Dr.Balaji P CHMC

7. Dr.Balaji P CHMC

8. Compound Microscope:
• Uses multiple lenses to get image from specimen
and uses another set of lenses to magnify it
before seeing it as a final image
• Advantages:
magnification is high
resolution is high
changeable objective lens
Dr.Balaji P CHMC

9. Dr.Balaji P CHMC

10. Lens
collection of
prisms as a unit
Dr.Balaji P CHMC

11. Magnification
degree of
enlargement of the
specimen’s image
by means of no.of
times of
length,breadth
Dr.Balaji P CHMC

12. Refraction
Bending of light when it
enters from one media
to another
Refractive Index
Ratio of velocity of light
in vacuum and in any
other medium
Dr.Balaji P CHMC

13. Resolution
Ability of a lens to
separate or
distinguish between
small objects that
are close together
Dr.Balaji P CHMC

14. Focal point & Focal length
Dr.Balaji P CHMC

15. Focal point
convergence of
light rays at a
point by lens
Focal length
Distance between
focal point and
centre of lens
Dr.Balaji P CHMC

16. Working distance
Distance between
the the front surface
of lens and the
surface of the cover
glass
Dr.Balaji P CHMC

17. Abbe’s formula:
• Described by a German physicist Ernst Abbe in 1870
• The resolution of a microscope depends upon the
numerical aperture of its condenser, objective lens,
and wave length of the light
• Goes by formula
d = 0.5λ
nsinθ
Dr.Balaji P CHMC

18. d- distance
λ - wave length of light
nsinθ - Numerical aperture
θ- angular aperture ( ½ the angle of cone of light enters
objective lens from specimen)
n – refractive index
Dr.Balaji P CHMC

19. NUMERICAL APERTURE
• Applies for condenser and objective lens
• Light gathering (converging) ability of a lens
• Depends on angular aperture(θ)
• Higher the numerical aperture lesser the
working distance and vice versa
• Cone of light depends on refractive index (n)
Dr.Balaji P CHMC

20. Dr.Balaji P CHMC

21. NA of various objectives:
4 X 0.1
10 X 0.25
40 X 0.65
100 X 1.25
Working Distance:
4 X 17-20 mm
10 X 4-8 mm
40 X 0.5-0.7 mm
100 X 0.1 mm
Dr.Balaji P CHMC

22. • Most microscopes posses NA 1.2 to 1.4
(objective lens)
• Condenser NA 0.9
• Refractive index (n) of air is 1
• Hence , lens working in air couldn’t give much
resolution, for which we are using immersion oil
which has more refractive index than air, which
in turn increases NA (max 1.25)
• Spectrum of light (blue green) used in
microscope is 380 nm -530 nm
Dr.Balaji P CHMC

23. • The maximum theoretical resolving power of a
microscope with an oil immersion objective with
blue-green light is approximately 0.2 mcm
• d= (0.5)(530 nm) = 0.212 / 0.2 mcm
1.25
• At best, a bright-field microscope can
distinguish between two dots around 0.2 mcm
apart
Dr.Balaji P CHMC

24. TYPES
• Bright field
• Dark field
• Phase contrast
• Differential interference contrast microscope
• Fluorescence microscopes
Dr.Balaji P CHMC

25. BRIGHT-FIELD MICROSCOPE
Dr.Balaji P CHMC

26. Dr.Balaji P CHMC

27. Optics of Light microscope
Light
Condenser
Sample
Objective lens
Focused at its focal length
Enlarged, inverted ,real image
Final image
Eye piece 1st lens
magnification further Eye piece 2nd lens
Dr.Balaji P CHMC

28. Optics of Light Microscope
Dr.Balaji P CHMC

29. Uses and advantages:
• Simple set up
• Used to view live / stained cells and organisms
• Little preparation is required
• Adaptable with new technology
Disadvantages:
• Biological specimens are of low contrast and
needs to be stained
• Needs stronger light source for high
magnification
• Artefacts of staining
Dr.Balaji P CHMC

30. Variants
• Inverted microscope
Dr.Balaji P CHMC

31. • Uses:
Live cell culture
Large specimens
Micromanipulation of sample
Advantages:
Large/ high weight specimens
More samples in shorter time
safety of objective
Works in same direction
Disdvantage:
Higher cost
Limited working distance
Thickness of container
Dr.Balaji P CHMC

32. • Stereo microscope
Dr.Balaji P CHMC

33. • Comparison microscope
Dr.Balaji P CHMC

34. • Petrographic microscope
Dr.Balaji P CHMC

35. Phase contrast Microscope
Dr.Balaji P CHMC

36. Phase contrast Microscope
• It works by principle of contrast enhancing
optical technique which produce high contrast
images of transparent specimens
• An annular diaphragm is used below the
condenser
• A phase plate is used above the objective lens
(within the lens tube)
• Annular stop, an opaque disk with a thin
transparent ring, which produces a hollow cone
of light
Dr.Balaji P CHMC

37. • As this cone passes through specimen ,some light
rays are bent and are retarded ¼ wave length
• This deviated light is focused to form an image of
the object
• Undeviated light rays strike phase ring in the phase
plate ,which is located in the objective
• after passing through phase plate ,undeviated light
is advanced by ¼ wavelength
• Finally , deviated and undeviated waves will be ½
wavelength out of phase and will cancel/merge each
other and forms final image
Dr.Balaji P CHMC

38. Dr.Balaji P CHMC

39. Dr.Balaji P CHMC

40. Dr.Balaji P CHMC

41. Dr.Balaji P CHMC

42. Dr.Balaji P CHMC

43. Optics of Phase contrast Microscope
Dr.Balaji P CHMC

44. Dr.Balaji P CHMC

45. Dr.Balaji P CHMC

46. Paramaecium Buccal cells Amoeba
Dr.Balaji P CHMC

47. Dr.Balaji P CHMC

48. Uses
• study of unstained living cells, microorganisms
such as bacteria,molds and their
shape,motility,subcellular particles bacterial
components such as endospores and inclusion
bodies etc.
Dr.Balaji P CHMC

49. Advantages:
• Observing of living cells in its natural state
• Specimen need not to be killed, fixed or stained
• High-contrast images
• Ideal for thin specimens
Disadvantages:
• Annuli or rings limit the aperture to some
extent, which decreases resolution
• Not ideal for thick specimens
• Images may appear grey or green, if white or
green lights are used, respectively, resulting in
poor photomicrography
• phase artifacts
Dr.Balaji P CHMC

50. Dr.Balaji P CHMC

51. Differential interference contrast
microscope
• Similar to the phase-contrast microscope
• But , a polarizer is used after light source
followed by a prism
• Two beams of plane polarized light at right
angles to each other generated by prism
• Object beam passes through the specimen, while
the reference beam passes through a clear area
of the slide
Dr.Balaji P CHMC

52. • After passing through specimen both waves will
combine and interfere with each others to form
image
• It creates an image by detecting differences in
refractive indices and thickness of specimen
• Finally a live, unstained specimen appears
brightly colored and three-dimensional
Dr.Balaji P CHMC

53. Optics of Differential-interference Phase
contrast Microscope
Dr.Balaji P CHMC

54. Dr.Balaji P CHMC

55. Uses
• Living cells and its structures such as cell walls,
endospores, granules, vacuoles,and eucaryotic
nuclei are clearly visible
• used to visualize living cells and for quantitative
studies
Dr.Balaji P CHMC

56. Advantages:
• It gives a better image than phase contrast
microscope
• 3D colorful images of specimen
• No Halo effect
• No shade off effect
• Disadvantages:
• three-dimensional image of a specimen
may not be accurate.
• The enhanced areas of light and shadow
might add distortion to the appearance of
the image.
Dr.Balaji P CHMC

57. Fungal spores
Paramecium
Dr.Balaji P CHMC

58. Egg of Trichuris trichiura Yeast cells
Dr.Balaji P CHMC

59. Dr.Balaji P CHMC

60. Dr.Balaji P CHMC

61. Dr.Balaji P CHMC

62. Dr.Balaji P CHMC

63. Dark field Microscopy
A special condenser lens is used with an opaque disc
at the centre, so that direct rays do not enter the
objective lens
Only light rays which are scattered by the specimen
enter the objective lens to form a bright image against
dark background
The field surrounding specimen appears black, while
the object itself is brightly illuminated
Its an illumintion technique used to enhance contrast
in unstained samples
Dr.Balaji P CHMC

64. Dr.Balaji P CHMC

65. Dr.Balaji P CHMC

66. Dr.Balaji P CHMC

67. • Variants
Treponema pallidum
Borrelia
Leptospira
Treponema Borrelia
Leptospira
Dr.Balaji P CHMC

68. Uses
Ideal for viewing objects that are unstained,
transparent and absorb little or no light
Live blood cells
Live bacteria
Other live organisms
Environmental water samples
Uses and advantages :
Dr.Balaji P CHMC

69. • Disadvantages: Prone to degradation,
distortion and inaccuracies
• A specimen that is not thin enough may appear
to have artifacts throughout the image
• Special care to be taken to prepare sample and
work place setup
• need to use oil on the condenser and/or slide , if
it contains liquid bubbles which will cause
images degradation, flare, distortion and even
decrease in contrast and details of the specimen.
• Dark field needs an intense amount of light to
work
Dr.Balaji P CHMC

70. References
• Prescot Microbiology 5th edition
• Bailey and Scott 14th edition
• Monica-Cheesbrough 2nd edition
Dr.Balaji P CHMC

71. Dr.Balaji P CHMC