1. Light Microscope
Terms and Practices

2. Objectives Describe: How thin sections are prepared and stained
The terms used in microscopy
The limits of resolution
Compare light and electron microscopy
Objectives

3. The light microscope depends on light passing through an object in order for it to be seen. The result is the sections must be very thin, less than 5 micrometers.
Light Microscope

4. The light microscope depends on light passing through an object in order for it to be seen. The result is the sections must be very thin, less than 5 micrometers. As a result, tissue preparation is most important.
Light Microscope

5. Fresh tissue can not be cut this thin because it will be crushed.
To avoid this the tissue is:
Dehydrated with alcohol to remove water
Light Microscope

6. Fresh tissue can not be cut this thin because it will be crushed.
To avoid this the tissue is:
Dehydrated with alcohol to remove water
Embedded with paraffin wax to support the tissue so it can be cut.
Light Microscope

7. Microtome

8. The Process

9. So why is this so important?
Everyday, tissue samples are taken from people and they need to be evaluated for disease processes like cancer.

10. Stains are used to enhance the tissue slide and to allow the histologist to identify various intracellular compounds.
Light Microscope

11. Oil Red O stains for neutral lipids
Light Microscope

12. Haematoxylin and eosin (H&E) staining is a very common stain
Haematoxylin and eosin (H&E) staining is a very common stain. Haematoxylin stains the nuclei blue and Eosin stains the cytoplasm pink.
Light Microscope

13. PAS staining is used to identify glycogen stored in tissues.
The light microscope

14. Compound light microscopes have two sets of lens which aid in the magnification of the specimen.
The objective lens are 4x, 10 x and 40 x
Magnification

15. Compound light microscopes have two sets of lens which aid in the magnification of the specimen.
The objective lens are 4x, 10 x and 40 x
The ocular lens is 10x
Magnification

16. Compound light microscopes have two sets of lens which aid in the magnification of the specimen.
The objective lens are 4x, 10 x and 40 x
The ocular lens is 10x
Total magnification is the product of the objective and ocular lens
Magnification

17. Scanning 4x objective

18. Low Power using 10 x objective

19. High Power using 40 x objective

20. Terms used for Microscopy
Resolution: The ability to distinguish 2 points as separate. The higher the resolution, the more detail is seen.
Terms used for Microscopy

22. Terms used for Microscopy
Depth of Field: The range of depth that can be focused in a specimen.
A thin depth of field will need to be continuously focused.
Terms used for Microscopy

23. Terms used for Microscopy
Depth of Field: The range of depth that can be focused in a specimen.
A thin depth of field will need to be continuously focused.
The higher the magnification, the more shallow the depth of field
Terms used for Microscopy

26. Terms used for Microscopy
Field of View: The area of the stage that can be viewed.
Terms used for Microscopy

27. Terms used for Microscopy
Field of View: The area of the stage that can be viewed.
The higher the magnification the smaller the field of view.
Terms used for Microscopy

29. Terms used for Microscopy
Working Distance: The distance between the objective lens and the microscope slide.
Terms used for Microscopy

30. Terms used for Microscopy
Working Distance: The distance between the objective lens and the microscope slide.
This decreases as magnification increases.
Terms used for Microscopy

32. Terms used for Microscopy
Contrast: Difference in the intensity between the object and the background. Stains aid in the contrast between the specimen and the background.
Terms used for Microscopy

34. Terms used for Microscopy
Illumination: This is the amount of light that is transmitted through the specimen.
The higher the magnification, the more light is required.
Terms used for Microscopy

35. Terms used for Microscopy
Numerical Aperture (NA): This is a complex concept.
Terms used for Microscopy

36. Terms used for Microscopy
Numerical Aperture (NA): This is a complex concept.
It is the ability to gather light and resolve a fine specimen at a fixed distance.
Terms used for Microscopy

37. Terms used for Microscopy
Numerical Aperture (NA): This is a complex concept. It is the ability to gather light and resolve a fine specimen at a fixed distance.
It takes in the quality of the lens, working distance, size of the lens and index of refraction.
Terms used for Microscopy

38. Terms used for Microscopy
Numerical Aperture (NA):
It is represented by the formula
NA= n x sin (a) where
NA is the numerical aperture
n is the refractive index
a is ½ of the opening angle
Terms used for Microscopy

40. Terms used for Microscopy
Numerical Aperture (NA) increases with lens magnification.
Terms used for Microscopy

41. Terms used for Microscopy
The limits of resolution & magnification
The greatest resolution with a light microscope is 0.2 microns.
The human eye alone has a resolution of 200 microns (0.2 mm)
Terms used for Microscopy

42. Terms used for Microscopy
The limits of resolution & magnification
Resolution is governed by the following relationship:
MR=l/NA of the objective & condenser
Terms used for Microscopy

43. Terms used for Microscopy
The limits of resolution & magnification
MR=l/NA of the objective & condenser
where l is the wavelength of light
Terms used for Microscopy

44. Terms used for Microscopy
The limits of resolution & magnification
MR=l/NA of the objective & condenser
where l is the wavelength of light
The shorter the wavelength, the greater the resolution.
Terms used for Microscopy

45. Terms used for Microscopy
The limits of resolution & magnification
The wave length of light ranges from
700 nm (red) to 400 nm (violet).
Using violet light the resolution of 0.2 microns is achieved.
Terms used for Microscopy

46. An electron microscope does not use light to illuminate a specimen.

47. An electron microscope does not use light to illuminate a specimen.
It uses the wave length of an electron which is about nm.
Electron Microscope

48. An electron microscope does not use light to illuminate a specimen.
It uses the wave length of an electron which is about nm.
Using the previous formula, a resolution of 0.1 nm or microns can be achieved.
Electron Microscope

49. Electron Microscope Magnifications are typically up to 1 million times
Cell ultrastructure is easily studied using this device.
Electron Microscope

50. This require specimens to be about 0.1micrometers in thickness
Electron Microscope

51. This require specimens to be about 0.1micrometers in thickness
Specimens must be embedded in plastic and stained with heavy metals like osmium tetroxide and uranyl acetate.
Electron Microscope

52. This require specimens to be about 0.1micrometers in thickness
Specimens must be embedded in plastic and stained with heavy metals like osmium tetroxide and uranyl acetate.
These will reflect electrons allowing an image to be formed.
Electron Microscope

53. Electron Microscope

54. Electron Microscope

55. Parts of a light microscope