Analysis of Cell Components

• Magnification is how much bigger the image is than the specimen

  magnification = image size/ actual size
• Resolution is how detailed the image is. More specifically, its how well a microscope distinguishes between two points that are close together.
• Transmission electron microscopes

  • TEMS use electromagnets to focus a beam of electron, which is then transmitted through the specimen
  • Denser parts of the specimen absorb more electrons, which makes them look darker on the image you end up with
  • TEMS are good because they give high resolution images so you see the internal structure of organelles
  • But you have to view the specimen in a vacuum, so they're no good for looking at living organisms
  • They can also only be used on thin specimens
• Scanning electron microscopes

  • SEMS scan a beam of electrons across the specimen.
  • This knocks off electrons from the specimen which are gathered in a cathode ray tube to form an image.
  • The images you end up with show the surface of the specimen and they can be 3D.
  • SEMs are good because they can be used on thick specimens, but they give lower resolution images
• Preparing microscope slide

  1. Start by pipetting a small drop of water onto the centre of the slide
  2. Then use tweezers to place a thin section of your specimen on top of the water drop
  3. Add a drop of stain
  4. Add a coverslip - stand the slip upright on the slide next to the water droplet and carefully lower it and try not to get any air bubbles
• Homogenisation - breaking up the cells

  • Homogenisation can be done in several different ways by vibrating the cells or by grinding up the cells in a blender. This breaks up the plasma membrane and releases the organelles into the solution
  • The solution must be kept ice cold to reduce the activity of enzymes that break down organelles
  • The solution should also be isotonic so it has the same concentration of chemicals as the cells being broken down to prevent the damage to the organelles through osmosis
  • A buffer solution should be added to maintain the pH
• Filtration

  • Next the homogenised cell solution is filtered through a gauze to separate any large cell debris or tissue debris,
  • The organelles are much smaller than the debris so they pass through the gauze
• Ultracentrifugation - separating the organelles

  1. The cell fragments are poured into a tube. The tube is put into a centrifuge and is spun at a low speed. The heaviest organelles like nuclei get flung to the bottom of the tube forming a thick sediment at the bottom - the pellet. The rest of the organelles stay suspended in the fluid above the sediment - the supernatant
  2. The supernatant is drained off, pure into another tube, and spun in the centrifuge at a higher speed. Again, the heaviest organelles form a pellet at the bottom of the tube. The supernatant containing the rest of the organelles is drained off and spun again in the centrifuge at an even higher speed
  3. This process is repeated at higher and higher speeds until all the organelles are separated out
  4. Order - nuclei, chloroplasts, mitochondria, lysosomes, ribosomes