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tHE lIGHT mICROSCOPE

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tHE lIGHT mICROSCOPE

  1. 1. Activity 1 Light Microscopy
  2. 2. Bright Field Microscope <ul><li>General Perspective </li></ul>
  3. 3. Schematic Perspective
  4. 5. Advantage <ul><li>Simplicity of setup </li></ul><ul><li>Allows viewing of live cells </li></ul><ul><li>Dark or highly colored </li></ul>
  5. 6. Specific Use <ul><li>Suited for utilization </li></ul><ul><li>Viewing stained or naturally pigmented specimen </li></ul><ul><li>Useless for some living specimens </li></ul>
  6. 7. Parts of Bright Field Microscope <ul><li>Base – supports the structure </li></ul><ul><li>Objective lenses- magnify the image. </li></ul><ul><li>Ocular - magnify the image from the objective lens. </li></ul>
  7. 8. <ul><li>Body Tube – send light to the ocular lens. </li></ul><ul><li>Condenser Lens- directs light to pass through the specimen. </li></ul><ul><li>Stage - platform that allows mechanical movement of a microscope slide. </li></ul>
  8. 9. <ul><li>Adjustment Knob – course and fine adjustment </li></ul><ul><li>Arm – support structure </li></ul><ul><li>Iris Diaphragm Lever- controls the amount of light entering the condenser and to specimen </li></ul><ul><li>Nosepiece – which are found the objectives. </li></ul><ul><li>Each objectives can be rotated into place by simply rotating the nosepiece </li></ul>
  9. 10. Dark Field Microscope <ul><li>General Perspective </li></ul>
  10. 11. Schematic Perspective
  11. 12. Accessory Parts
  12. 13. Advantage <ul><li>Quality of image is impressive </li></ul><ul><li>Raised features </li></ul>
  13. 14. Specific Use <ul><li>Live and biological samples </li></ul><ul><li>Study of crystals and crystal defects </li></ul>
  14. 15. Phase Contrast Microscope <ul><li>General Perspective </li></ul>
  15. 16. Schematic Perspective
  16. 17. Accessory Parts
  17. 18. Advantage <ul><li>Does not require staining </li></ul><ul><li>living cells can be examined in their natural state without being killed, fixed, and stained </li></ul>
  18. 19. Specific Use <ul><li>Transparent specimens </li></ul><ul><li>Cell parts are transparent </li></ul>
  19. 20. Fluorescent Microscope <ul><li>General Perspective </li></ul>
  20. 21. Schematic Perspective
  21. 22. Accessory Parts
  22. 23. Advantage <ul><li>Detect structures and molecules within the cell </li></ul>
  23. 24. Specific Use <ul><li>Living cells and internal components are contrasted against the background giving greater definition and detail of cell structure </li></ul><ul><li>Cells need not be fixed or stained </li></ul><ul><li>Absorb light at one wavelength and emit light at another </li></ul>
  24. 25. Differential Interference Contrast Microscope General Perspective
  25. 26. Light Pathway in DIC Microscope
  26. 27. Inverted micoscope <ul><li>General Perspective </li></ul>
  27. 28. Light pathways of Inverted Microscope
  28. 29. Dissecting Microscope
  29. 30. Pathway of Light
  30. 31. Stereo Microscope <ul><li>General Perspective </li></ul>
  31. 32. Schematic Perspective
  32. 33. Sample illumination is via transmitted white light . Simplicity of setup with only basic equipment required. No sample preparation required, allowing viewing of live cells Bright Field Microscope Specific Use Advantage Types of Light Microscope
  33. 34. Transparent specimens Cell parts are transparent Does not require staining living cells can be examined in their natural state Phase Contrast Microscope Live and biological samples Study of crystals and crystal defects Quality of image is impressive Raised features Dark field Microscope
  34. 35. Living cells and internal components are contrasted against the background giving greater definition and detail of cell structure Detect structures and molecules within the cell Fluorescent Microscope
  35. 36. Used for dissection to get a better look at the larger specimen. Gives true color with 20x and 40x power using 10x eyepieces and rotating turret of 2x and 4x objectives Dissecting Microscope Allow the specimen to remain active for long periods. Allow to observe a living cells or organisms at the bottom of a large container Inverted Microscope Allows the specimen to be seen more clearly Do not mask or otherwise obstruct the objective and condenser apertures Differential Interference Contrast Microscope
  36. 37. designed long working distance objectives & wide field eyepieces produce an extremely large, brilliant & bright images. Light in weight, fixed achromatic objectives 2x or 3x Stereo Microscope
  37. 38. Formulas <ul><li>R = (0.61 x λ) / N.A. </li></ul><ul><li>λ – Light Wavelength </li></ul><ul><li>N.A. – Numerical Aperture </li></ul>
  38. 39. Wavelength of ROYGBIV
  39. 40. Numerical Aperture <ul><li>N.A. = n sin θ </li></ul><ul><ul><li>n : the refractive index of the media at d-line (587m) </li></ul></ul><ul><ul><li>for dry objective n = 1.000 air </li></ul></ul><ul><ul><li>for oil objective n = 1.515 oil </li></ul></ul><ul><ul><li>for water objective n = 1.333 water </li></ul></ul><ul><ul><li>θ = half angle of incident rays to the top lens of the objective. </li></ul></ul>
  40. 41. 2.14 - 2.20 Cubic Zirconia 2.41 Diamond 1.73-1.89 Garnet 1.62 Tourmaline 1.54 Salt 1.54 Quartz 1.43 Fluorite 1.36 Ethyl alcohol 1.33 Water 1.31 Ice 1.000293 Air Refractive Index Substance
  41. 42. Steps in Preparing the Specimen <ul><li>Fixation – it is done by putting chemicals that preserve material in a lifelike condition, thus, the specimen can’t be distorted </li></ul><ul><li>Dehydration – water is removed from the specimen using ethanol </li></ul><ul><li>Staining – most of biological material is transparent and needs staining to increase the contrast between different structures </li></ul>
  42. 43. <ul><li>Mounting – mounting on the slide protects the material so that it is suitable for viewing over a long period. </li></ul>
  43. 44. Problems Encountered and Corrective Measures <ul><li>When using compound microscope and you come with very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings or the so called airy-disc. </li></ul><ul><li>Limitations of lens design which can result in increased magnification without increased resolution resulting to image that is larger but not clearer and could not present more detailed information. </li></ul><ul><li> Two objects must be 0.1 mm apart so that they will be perceived as two, there are lens deign that even though objects appears 0.1 mm apart, the edges becomes blurry that we detect two objects as single. </li></ul>
  44. 45. Hypothetical Problems <ul><li>Using the oil immersion objective of a bright-field microscope, an object is seen and measured as 1.2cm long. What is the actual size of the observed object as expressed in nanometers? </li></ul>
  45. 46. <ul><li>1.2cm = y </li></ul><ul><li>1000cm 1y </li></ul><ul><li>y= 1.2cm x 1y = 0.0012 cm </li></ul><ul><li>1000cm </li></ul>
  46. 47. References <ul><li>Davidson, M. & Abramowitz, M. 2003. Brightfield Microscopy Digital Image Gallery . MolecularExpressions, http://micro.magnet.fsu.edu/primer/anatomy/brightfieldgallery/index.html . </li></ul><ul><li>Caprette, D.2005. Bright Field Microscopy . Experimental Biosciences, http://www.ruf.rice.edu/~bioslabs/bios211/index.html. </li></ul><ul><li>P. Hirsch, A. Howie, R. Nicholson, D. W. Pashley and M. J. Whelan.2008. Dark field microscopy . Wikipedia the free encyclopedia, http://en.wikipedia.org/wiki/Dark_field_microscopy . </li></ul><ul><li>Douglas B. Murphy, Ron Oldfield.2003. Principles of phase contrast microscopy. MicroscopyU, http://www.microscopyu.com/articles/phasecontrast/phasehome.html . </li></ul><ul><li>Frängsmyr,T. & Ekspång,G.1993. The Fluorescence Microscope- Preparation of a Specimen .Nobelprize,http://nobelprize.org/educational_games/physics/microscopes/fluorescence/preparation.html </li></ul>

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