Analysis of Cell Components


Magnification and Resolution

  • Magnification is how much bigger the image is than the specimen
    • Magnification = image/object
  • Resolution is how detailed the image is, or how wel the mcroscope distinguishes between two points that are close together
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Optical Microscopes

  • They use light to form an image (longer wavelength)
  • Maximum resolution of about 0.2 micrometres
    • Not able to see ribosomes, endoplasmic reticulum and lysosomes. Possible to see mitochondria but not in great detail
    • Maximum useful magnification is about x 1500
  • Viewing specimens using slides:
    • Pipette a small drop of water onto the slide, then use tweezers to place a thin section of your specimen on top of the water drop
    • Add a drop of a stain. Stains are used to highlight objects in a cell. For example, eosinis used to make the cytoplasm show up. Iodine in potassium iodide solution is used to stain starch grains
    • Finally, add a cover slip. To do so, stand the slip upright on the slide, next to the water droplet. Then carefully tilt and lower it so it covers the specimen. Try not to get any air bubbles under it as they will obstruct the view of your specimen
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Electron Microscopes

  • They use electrons to form an image
  • They have a higher resolution and give a more detailed image. Their maximum resolution is about 0.0002 micrometres
  • The maximum useful magnification of an electron microscope is about x 1 500 000
    • TEM
      • Use electromagnes to focus a beam of electrons, which is then transmitted through the specimen
      • Denser parts of the specimen absorb more electrons, which makes them look darker
      • Give high resolution images, so you see the internal structure of organelles, but they can only be used on thin specimens
    • SEM
      • Scan a beam of electrns across the specimen. This knocks off electrons from the specimen, which are gathered by a cathode ray tube to form an image
      • They show the surface of the specimen and can be 3-D
      • They can be used on thick specimens, but have a lower resolution
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Cell Fractionation

  • Homogenisation
    • Grind the cells up in a blender or vibrate the cells vigorously. This breaks up the plasma membrane. The solution must be ice-cold to reduce the activity of enxymes that break down organelles. It should also be isotonic to prevent damage to organelles through osmosis. It should also have a buffer solution to maintain the pH
  • Filtration
    • The homogenised cell solution is filtered through a gauze to seperate any large cell debris, like connective tissue, from the organelles
  • Ultracentrifugation
    • The cell fragments are poured into a tube. This tube is placed in a centrifuge and spun at a low speed. The heaviest organelles get flung to the bottom of the tube by the centrifuge. They form a thick sediment at the bottom called the pellet. The rest of the organelles stay suspended in the fluid above the sediment - the supernatant
    • The supernatant is drained off, poured into another tube and spun again at a high speed, for a longer time. The cycle continues until the organelles are all seperated out, or you get the organelles you want
    • The order goes like this: nuclei, chloroplasts, mitochondria, lysosomes, endoplasmic reticulum, then the ribosomes
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