Microscopy and properties of lights

1. The Microscope
Mentor: Dr. Umesh Kumar
Professor & Head,
Department of Physiology
Government Medical College, Kota
By: Dr Shailendra Tyagi
PG-Resident,
Department of Physiology
Government Medical College,
Kota

2. Lerning objective:
❖ Light
○ Nature of Light
○ Dimensions of Light
○ Properties of Light
❖ Human Eye
❖ The Microscope
○ Principle of Microscopy
○ History of Microscopy
○ Types of Microscope
○ Terms related to Microscope
❖ Image Formation
❖ Focusing of Microscope
❖ Uses of Microscope
❖ Handling and Care of Microscope

3. Light

4. Nature of Light
● Light is electromagnetic radiation.
● The visible spectrum of this radiation with wavelengths between 400nm and 700nm.
● The elementary particle that defines light is the photon.
● Light shows dual nature:
i.Partical nature
ii.Wave nature
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5. i.Partical Nature: illustrated by the photoelectric
effect.
Photoelectric effect: Discovered by Einstein in
1905.
E=hf=hc/λ
● The number of ejected electron is directly related to
intensity of applied light.
● The particle or ray model of light is illustrated by the
properties of reflection and refraction.
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6. ii.Wave Nature: illustrated by Young’s Double slit experiment.
● Discovered by Thomas Young in 1801.
● The wave model of light is illustrated by the properties of reflection, refraction,
diffraction, interference, and polarization.
Types of waves:
i.Transverse- electromagnetic waves.
ii.Longitudinal- sound waves.

7. Amplitude(Intensity):
● Maximum displacement of wave from an
equilibrium position.
Wavelength:
● It is the distance between two wave crest.
Dimensions of Light
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8. Frequency:
● It refer to the number of times a wave crest
passes particular point in one second.
● Frequency remain constant for a light wave.
Phase:
The phase involves the relationship between
the position of the amplitude crests and
troughs of two waveforms

9. Terms Related to Light
Radiation: way to transfer of energy in the form of a wave.
Electromagnetic Radiation: also known as Light. Transfer energy and information
from one place to another in form of coupled electric and magnetic waves.
Visible Spectrum: the range of electromagnetic spectrum that the human eye perceive.
Ranges from 400nm-700nm.
Speed of Light:The wave speed of all types of light in a vacuum is called the speed of
light.(c = 300,000 km/sec.)
● The speed of the wave is related to the frequency and wavelength of the wave.
Wave speed = frequency x wavelength
● Light waves require NO material medium to travel from place to place.

10. Properties of Light

11. Effect of material on Light:
Transparent material: Transmit Light.
Translucent material: Scatteres and Transmit
Light.
Opaque material: Absorb and Reflect Light.
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12. Reflection: The light bouncing back after falling on
an object.
Law of Reflection:The angle of lncidence is
equal to angle of Reflection.
Regular Reflection:Through smooth surface.
Diffuse Reflection: Through rough surface.
Total Internal Reflection: Complete
reflection of a ray of light within a medium. Occurs if the
angle of incidence is greater than a certain limiting angle,
called the critical angle
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13. Refraction: Direction change of a ray of
light passing from one transparent medium to
another with different optical density.
Index of Refraction: It is the amount of
Light refract in a medium.
Dispersion: Separation of light into its
constituent wavelengths when entering a
transparent medium.
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14. Absorption: When light passes through an object the
intensity is reduced depending upon the wavelength
absorbed. Thus the selective absorption of white light
produces colored light.
Transmission: Moving of light through a material.
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15. Interference is the interaction of the two waves.
Constructive interference=reinforcing interaction.
Destructive interference=canceling interaction.
Superposition is the method used to model the
composite form of the resulting wave.
Diffraction:Light rays bend around edges - new
wavefronts are generated at sharp edges.
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16. Scattering of Light: It is the phenomenon of bouncing off
electromagnetic radiation by the atoms or molecules of the medium through
which they are travelling.
● Elastic Scattering: If the energy of the scattered light is conserved.
● Inelastic Scattering: Energy of the scattered light is not conserved.
a=AV/rλ2
where
A is the amplitude of the incident light
λ is the wavelength of the incident light
a is the amplitude of the scattered light
r is the distance from the scattering particle
● Intensity of the scattered light (Is) varies directly as the square of the
amplitude (a2) of the scattered light. So
Is∝1λ4
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17. Polarisation: is the process of converting non-polarized light into
polarised light.
The light in which particles vibrate in all various planes is known as
unpolarised light.
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18. Methods Used in the Polarisation of Light:
● Polarisation by Polaroids
● Polarization by Scattering
● Polarisation by Reflection and Refraction

19. The Human Eye

20. Cornea: The transparent part of the eye that covers the iris and the pupil and
allows light to enter the inside.
Resolving power of Cornea is 40D-42D.
Crystalline lens:Biconvex, transparent, avascular structure.
Resolving power of Lens is 18D-20D.
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21. Retina:layer of nervous tissue that covers the inside of the back two-thirds of the
eyeball.
● The primary light-sensing cells in the retina are the photoreceptor cells known as rods
and cones.
● Blind spot: area where optic nerve passes. There are absence of photoreceptor cells.
● Fovea centralis: Area where maximum photoreceptor cells present. Adapted for high-
acuity vision.
Visual Acuity:The ability of the eye to recognize two very closely situated points of
light or lines.
Refractive power of human eye- 60D.
Resolving power of human eye- 0.25mm.

22. The Microscope

23. Working principle of microscope:
● When the light source illuminates the object.
● The first image of the object
is formed by objective lens which is a real,
inverted and magnified image.
● The image formed by the objective functions
as the object for eyepiece, which produces the
final,virtual and magnified image.

24. History

25. 1595: Hans and Zacharias Janssen
● Created the first microscope.
● Which was composed of two lenses in a tube.
1665:Robert Hook
● Invented the term “cell”
● Used microscope to study cells
● Write book “Micrographia”
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26. 1674: Anton van Leeuwenhoek
● Created the microscope that was capable of magnifying
object over 300x.
● First person to observe movement of single cell
organisms.
1729: Chester Moore Hall
● Created the first Acromatic lenses.
● Created first refracting telescope free from colour
distortion.
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27. 1830:Joseph Jackson Lister
● Improved image resolution by correcting spherical
aberrations.
● Determined the true form of red blood cells in
mammals.
1873:Ernst Abbe
● Invented apocromatic lens system to eliminate light
distortion.
● Discovered an optical formula called the “Abbe sine
condition”.
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28. 1903:Richard Zsigmondy
● Invented ultra microscope to study colloids.
1931: Ernst Ruska
● Designed the electron microscope.
1932: Frits Xernike's
● Invented phase-contrast microscope.
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29. 1938:James Hillier and Albert Prebus
● Built the first successful high resolution
microscope.
1981:Gerd Binnig Heinrich Rohrer
● Invented scanning tunneling microscope.
1986: Gerd Binning Quate and Gerber
● Invented the Atomic force microscope (AFM).
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30. 1988:Kingo Itaya
● Invented the Electrochemical scanning tunneling
microscope.
1991: Kelvin
● Invented probe force microscope.
2009:Pratibha Gai
● Invented the in-situ atomic resolution environmental
transmission Electron Microscope.

31. Types of Microscope
1. Binocular Microscope.
2. Dissection Microscope.
3.Dark-field Microscope.
4. Phase-contrast Microscope.
5. Interference-contrast Microscope.
6. Polarizing Microscope.
7. Fluorescence Microscope.
8. Transmission Electron Microscope (TEM).
9. Scanning electron microscope (SEM).

32. Terms Related to Microscope

33. Magnification:
The power of the microscope to enlarge the image of an object.
M= mo×me
Objective lens Ocular lens Total magnification
Scanning 4X 10X 40X
Low power 10X 10X 100X
High power 40X 10X 400X
Oil Emerson 100X 10X 1000X
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34. Resolution
● The power of objective lens to differentiate two distinct point clearly.
Limit of resolution:
● The minimal distance of two points that can be distinguished as separate objects.
D=0.61λ/n sin α
Where
D= limit of resolution
λ = wavelength of light used
n= refractive index
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35. Numerical aperture:
● The angle between sample and objective lens made
by light rays is called as numerical aperture.
● The higher the numerical aperture of a lens, the
better the resolution.
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36. Aberrasion:
A.Chromatic aberrasion
Correction aberrasion:
Acromatic objective and apocromatic objectives.
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37. B.Spherical aberrasion :
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38. Oil immersion:
● The white light used in a compound light microscope has relatively long wave
length and cannot resolve structures smaller than about 0.2 μm.
● Immersion oil is placed between the glass and
objective lens.
● The oil enhances the resolution by preventing light rays from dispersing and
changing wave length after passing through the specimens.
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39. The Effect of immersion oil on resolution:
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40. Working Distance:
● The working distance is the distance between the
objective and the slide under study.
● This distance decreases with increasing magnification.
Field of View :
● The field of view is the diameter of the circle of light that you see when
looking into a microscope.
● The lowest powers have higher field of view.
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41. Depth of Field :
● Low magnification objectives have more depth of field than high
magnification objectives.
Depth of Focus:
● It is just the reverse of the depth of field, where here greater depth of focus
occurs with high magnification objectives.
Flatness of Field:
● A quality describing the appearance of the field of view as being flat from
edge-to-edge.

42. Image Formation

43. Simple Microscope:
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44. Compound Microscope:
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45. Image Formation:
● Objective forms a real, inverted, and enlarged
image in the upper part of the body tube.
● The eyepiece collects the divergent rays of light of
the primary image and passes these through the eye
lens,therefore the image seen by the eye is
virtual, inverted, and magnified.

46. Part's of Microscope:
1. Mechanical supporting system
2. Optical system
3. Illumination system
4. Adjustment system
5. Other
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47. 1.Mechanical supporting system:
• Nosepiece:Holds the objective lenses,Rotates to enable magnification.
• Arm:Supports the upper parts of the microscope.
• Base:Supports the whole microscope.
• Stage:Supports the slide
2.Optical system:
● Eyepiece(ocular): - usually 10x.
● Objective Lenses: usually 10x-100x
● Scanner: usually 4x
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48. 3.Illumination system:
● Illumination is the application of light onto an object or specimen in a microscope.
● The illuminator = source of light.
● Light Source:Provides light necessary for viewing the specimen.
● Diaphragm/Iris:Wheel or lever located below the stage opening.
● Conndensor: focus the light on the specimen.
● Filter: Blue or white filter below condensor.
● Mirror:Reflect light rays from light source to object.
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49. 4.Adjustment system:
● Coarse Adjustment Knob:Used only when focusing the low power (4x) objective lens.
● Fine Adjustment Knob:Used when focusing the medium power (10x) and high power
(40x) objective lenses.
5.Other:
● Power switch
● Stage clips
● Stage stop
● Stage Adjustment
● Condensor Adjustment

50. Focusing of Microscope

51. Low power:
● Put the slide on the stage and bring specimen over central aperture.
● Lower the condenser and partly close the diaphrag
● Focus the object using coarse adjustment
● Use fine adjustment as required
High power:
● Rotate the nose piece to high power.
● Adjust the condenser and open the diaphragm to admit enough light.
● Use fine adjustment as required.
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52. Oil immersion:
● Focus the object first under low power.
● Rotate nose piece to oil immersion objective.
● Raise the condenser completely.
● Open the diaphragm.
● Keep a drop of immersion oil.
● Use fine adjustment as required.

53. Uses of microscope:
● Education
● Medical
● Industry
● Science
● Judiciary

54. Care of Microscope:
● Transport: When you pick up the microscope and walk with it, grab the
arm with one hand and place your other hand on the bottom of the base.
● Cleaning of lens: Never touch the lenses with your fingers. Use only
LENS PAPER to clean the glass.
● Storage: When you are finished with your "scope" assignment, rotate the
nosepiece so that it's on the low power objective, roll the nosepiece so that it's
all the way down to the stage, then replace the dust cover.
● Cleanup: Clean all slides, materials, and work area when you're done.