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Microscopy
1
322 Histological Techniques
Agenda for today
 What is Microscope, Facts and structures ?
 What is it use for?
 How is it function?
 What type of Microscope?
2
3
Fact About Microscopes
4
Light microsocopes are used to see details, and enlarged
images of small objects.
Magnification is enlarging an image
Resolution is the amount of fine detail that can be seen
Light is focused onto the specimen (i.e. the histology slide) by
a condenser.
About Microscopes con.
5
The image produced is magnified by a combination of the
objective lens and the eyepiece lens.
Usually, the eyepiece lens gives a x10 magnification.
Three objective lenses are usually used: x10, x40 and x100
The x100 lens is usually an oil-immersion lens - you need to
view the sample through a drop of oil.
Disadvantage of Light Microscopy
 The resolving power is limited:
The resolving power (resolution) of a light microscope is.
Resolution = 0.61 x lambda /NA
Lambda is the wavelength of the illuminating radiation,
NA the numerical aperture of the lens.
For a light microscope, the highest practicable NA is around 1.4. For white
light (lambda is approximately 0.53 m, the resolving power is 0.231 m, or
231nm.
Under optimal conditions, with a high numerical aperture lens, you can only
resolve, or see as separate particles, two particles that are more than 200 nm
apart.
6
Lenses and the Bending of Light
7
 light is refracted (bent) when passing from
one medium to another
 refractive index
 a measure of how greatly a substance slows the
velocity of light
 direction and magnitude of bending is
determined by the refractive indexes of the
two media forming the interface
Lenses
8
 focus light rays at a specific place called the focal point
 Focal length is the distance between center of lens and
focal point.
 The strength of lens related to focal length
 short focal length more magnification
9
The Light Microscope
10
 many types
 bright-field microscope
 dark-field microscope
 phase-contrast microscope
 fluorescence microscopes
 are compound microscopes
 image formed by action of 2 lenses
The Bright-Field Microscope
11
 produces a dark image against a brighter background
 has several objective lenses
 parfocal microscopes remain in focus when objectives are
changed
 total magnification
 product of the magnifications of the ocular lens and the
objective lens
12
13
Microscope Resolution
14
 Ability of a lens to separate or distinguish small objects
that are close together
 Wavelength of light used is major factor in resolution
shorter wavelength  greater resolution
15
16
working distance
— distance between the front surface of lens and surface of
cover glass or specimen
17
The Dark-Field Microscope
18
 Produces a bright image of the object against a dark
background
 Used to observe living, unstained preparations
19
The Phase-Contrast Microscope
20
 Enhances the contrast between intracellular
structures having slight differences in refractive index
 Excellent way to observe living cells
21
22
The Differential Interference Contrast
Microscope
23
 Creates image by detecting differences in refractive
indices and thickness of different parts of specimen
 Excellent way to observe living cells
The Fluorescence Microscope
24
 Exposes specimen to ultraviolet, violet, or blue light
 Specimens usually stained with fluorochromes
 Shows a bright image of the object resulting from the
fluorescent light emitted by the specimen
25
26
Newer Techniques in Microscopy
 confocal microscopy
and scanning probe
microscopy
 have extremely high
resolution
 can be used to observe
individual atoms
27
Confocal Microscopy
28
 confocal scanning laser microscope
 laser beam used to illuminate spots on specimen
 computer compiles images created from each point to
generate a 3-dimensional image
29
30
Scanning Probe Microscopy
31
 Scanning tunneling microscope
 steady current (tunneling current) maintained between
microscope probe and specimen
 up and down movement of probe as it maintains current is
detected and used to create image of surface of specimen
Scanning Probe Microscopy
32
 Atomic force microscope
 sharp probe moves over surface of specimen at constant
distance
 up and down movement of probe as it maintains constant
distance is detected and used to create image

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Microscope of ppt for botany major this is a project

  • 2. Agenda for today  What is Microscope, Facts and structures ?  What is it use for?  How is it function?  What type of Microscope? 2
  • 3. 3
  • 4. Fact About Microscopes 4 Light microsocopes are used to see details, and enlarged images of small objects. Magnification is enlarging an image Resolution is the amount of fine detail that can be seen Light is focused onto the specimen (i.e. the histology slide) by a condenser.
  • 5. About Microscopes con. 5 The image produced is magnified by a combination of the objective lens and the eyepiece lens. Usually, the eyepiece lens gives a x10 magnification. Three objective lenses are usually used: x10, x40 and x100 The x100 lens is usually an oil-immersion lens - you need to view the sample through a drop of oil.
  • 6. Disadvantage of Light Microscopy  The resolving power is limited: The resolving power (resolution) of a light microscope is. Resolution = 0.61 x lambda /NA Lambda is the wavelength of the illuminating radiation, NA the numerical aperture of the lens. For a light microscope, the highest practicable NA is around 1.4. For white light (lambda is approximately 0.53 m, the resolving power is 0.231 m, or 231nm. Under optimal conditions, with a high numerical aperture lens, you can only resolve, or see as separate particles, two particles that are more than 200 nm apart. 6
  • 7. Lenses and the Bending of Light 7  light is refracted (bent) when passing from one medium to another  refractive index  a measure of how greatly a substance slows the velocity of light  direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface
  • 8. Lenses 8  focus light rays at a specific place called the focal point  Focal length is the distance between center of lens and focal point.  The strength of lens related to focal length  short focal length more magnification
  • 9. 9
  • 10. The Light Microscope 10  many types  bright-field microscope  dark-field microscope  phase-contrast microscope  fluorescence microscopes  are compound microscopes  image formed by action of 2 lenses
  • 11. The Bright-Field Microscope 11  produces a dark image against a brighter background  has several objective lenses  parfocal microscopes remain in focus when objectives are changed  total magnification  product of the magnifications of the ocular lens and the objective lens
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  • 13. 13
  • 14. Microscope Resolution 14  Ability of a lens to separate or distinguish small objects that are close together  Wavelength of light used is major factor in resolution shorter wavelength  greater resolution
  • 15. 15
  • 16. 16 working distance — distance between the front surface of lens and surface of cover glass or specimen
  • 17. 17
  • 18. The Dark-Field Microscope 18  Produces a bright image of the object against a dark background  Used to observe living, unstained preparations
  • 19. 19
  • 20. The Phase-Contrast Microscope 20  Enhances the contrast between intracellular structures having slight differences in refractive index  Excellent way to observe living cells
  • 21. 21
  • 22. 22
  • 23. The Differential Interference Contrast Microscope 23  Creates image by detecting differences in refractive indices and thickness of different parts of specimen  Excellent way to observe living cells
  • 24. The Fluorescence Microscope 24  Exposes specimen to ultraviolet, violet, or blue light  Specimens usually stained with fluorochromes  Shows a bright image of the object resulting from the fluorescent light emitted by the specimen
  • 25. 25
  • 26. 26
  • 27. Newer Techniques in Microscopy  confocal microscopy and scanning probe microscopy  have extremely high resolution  can be used to observe individual atoms 27
  • 28. Confocal Microscopy 28  confocal scanning laser microscope  laser beam used to illuminate spots on specimen  computer compiles images created from each point to generate a 3-dimensional image
  • 29. 29
  • 30. 30
  • 31. Scanning Probe Microscopy 31  Scanning tunneling microscope  steady current (tunneling current) maintained between microscope probe and specimen  up and down movement of probe as it maintains current is detected and used to create image of surface of specimen
  • 32. Scanning Probe Microscopy 32  Atomic force microscope  sharp probe moves over surface of specimen at constant distance  up and down movement of probe as it maintains constant distance is detected and used to create image