Microscopy

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Microscopy

  1. 1. MICROSCOPY Dr.Riyaz
  2. 2. History of Microscopy • Born in Netherlands • Fabric Merchant • Surveyor • Wine Assayer • Minor City Official. • Learnt to grind lenses and started building simple microscopes by 1668 ANTONIE VAN LEEUWENHOEK
  3. 3. Animalcules
  4. 4. Common terms in Microscopy • Aberration  Unwanted Artifacts – Dust or Oil on Optical Surfaces – Improper alignment – Improper aperture settings – Imperfections in lens system • Spherical aberration – Image in focus I n the centre and blurred at the periphery • Chromatic aberration – Colour fringes within image field
  5. 5. Spherical aberration
  6. 6. Chromatic aberration
  7. 7. Contrast Difference in image luminance
  8. 8. Depth of field • Vertical distance between the nearest and farthest objects in specimen that appear to be in sharp focus. • Depth of field increases with numerical aperture of lens
  9. 9. Immersion fluid ( Oil ) • Any liquid that occupies space between the object and microscope objective lens • Required for objectives that have working distance of 3mm or less • Immersion fluid should have the same refractive index as that of slide (1.515) • Immersion fluid maximizes effective numerical aperture of objective lens • Minimizes light refraction
  10. 10. 40X
  11. 11. 100X
  12. 12. SEM
  13. 13. What else?
  14. 14. Numerical Aperture • Measure of light gathering capacity of Lens or Condenser Objective Mirror Condense r position Iris Diaphrag m Magnification Objective Eye Total Low Concave Lowest Partially closed 10 10 100 High power Concave Slightly raised Partially open 45 10 450 Oil immersio n Plane Fully raised Fully open 100 10 1000
  15. 15. Dissection Microscopes
  16. 16. I M.B.B.S
  17. 17. • 1590 by Dutch spectacle makers • OBJECTIVES – 4X – 10X – 40X – 100X
  18. 18. Differences • Field Diaphragm – Light source • Substage condenser – Gathers light and concentrates into a cone onto the specimen
  19. 19. How images are formed?
  20. 20. DARK FIELD MICROSCOPY
  21. 21. DARK FIELD MICROSCOPE
  22. 22. • Specialized illumination technique • View thin organisms like spirochetes & leptospira
  23. 23. Special Dark field condenser BRIGHT FIELD DARK FIELD
  24. 24. • BLOCKS CENTRAL PATH OF LIGHT & DIRECTS LIGHT AWAY FROM OBJECTIVE AT AN OBLIQUE ANGLE
  25. 25. • Bacteria have a refractive index slightly different from the surrounding. • Light passing through the bacteria enters objective • Light not passing through the bacteria does not enter objective • So , end result – Bright organisms against dark background
  26. 26. Phase contrast microscopy
  27. 27. Phase contrast microscope
  28. 28. Why Phase Contrast? • Many unstained biological specimens are virtually transparent under bright field illumination • Visibility can be improved in wet mount and cell cultures by reducing the opening size of substage condenser and iris diaphragm Loss of resolution. Artifacts • Phase contrast improves the contrast in these specimens without loss in resolution
  29. 29. Working principle.. • The phase contrast microscope uses the fact that the light passing trough a transparent part of the specimen travels slower and, due to this is shifted compared to the uninfluenced light. • This difference in phase is not visible to the human eye. • However, the change in phase can be increased to half a wavelength by a transparent phase-plate in the microscope and thereby causing a difference in brightness. • This makes the transparent object shine out in contrast to its surroundings.
  30. 30. Working principle..
  31. 31. • When dealing with transparent and colorless components in a cell, dyeing is an alternative but at the same time stops all processes in it. The phase contrast microscope has made it possible to study living cells, and cell division is an example of a process that has been examined in detail with it. • The phase contrast microscope was awarded with the Nobel Prize in Physics, 1953.

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