microscopes, magnification, and cell fractionation

• resolution = the ability to distinguish between objects close together
• Optical microscopes

  • Uses glass lenses to focuses light • Views both dead and living specimens
  • In colour.
  • As light is used, light has a longer wavelength than electrons, so the light microscope has a lower resolution compared to the electron microscopes.
• Electron microscope

  • Uses electrons pass through / enter (thin) specimen and focuses using magnets.

  • Views dead samples as the microscopes must be in a vacuum.

  • In black and white.

  The denser parts absorb more electrons so appear darker. Electrons have short wavelength so give high resolution, so smaller organelles can be seen and with greater detail.
• Limitations of electron microscopes:

  1. The preparation of specimen could lead to the production of artefacts
  2. Complex staining method / complex / long preparation time;
• There are 2 types of electron microscopes:

  Transmission electron microscopes- View 2D images but can view inside the cell as specimens are very thin.

  Scanning electron microscope- Thin sections do not need to be prepared. The SEM shows surface of specimen and a 3-D image is formed.
• 1. Cell homogenisation to break open cells.- often with a blender
  2. Filter to remove (large) debris / whole cells.
  3. Use isotonic to maintain the water potential of the solution to prevent osmosis so the organelles do not shrivel or burst.
  4. Ice cold to reduce kinetic energy of enzymes which could damage the organelles.
  5. Use a buffer to keep the pH constant to prevent protein/enzyme denaturation.
  6. Centrifuge (at lower speed / 1000 g) to separate nuclei / cell fragments / heavy organelles.
  7. Re-spin (supernatant / after nuclei / pellet removed) at higher speed to get mitochondria in pellet
• highest to lowest density:

  • nucleus
  • mitochondria / chloroplast
  • RER, SER + golgi
  • Ribosomes