3.2.1.3 Methods

• Magnification = image size/actual size
• Optical microscope

  Few organelles can be viewed (too small/thin to be resolved)

  Lowe magnification

  Lower resolution due to longer wavelength of light

  Live specimens can be viewed

  Less distortion
• Transmission microscope

  Difficult preparation of specimen

  Thin section needed - risk of damaging specimen

  Introduction of artefacts into the specimen

  Unable to see live specimens

  Position of organelles can vary - different organelles in different sections can be viewed

  Higher magnification

  Higher resolution
• Scanning microscope 


  Same limitations as transmission microscope except specimen doesn't need to be thin as electrons do not penetrate it

  3D image is viewed

  Lower resolution and magnification than transmission but still higher than optical
• Cell fractionation

  1. Intact cells are suspended in an ice cold (enzyme activity low to prevent digestion of organelles), buffered (keep pH constant) sucrose (prevent osmosis and lysis) solution
  2. Homogeniser breaks open cell surface membrane to release organelles
  3. Homogenate contains membrane bound organelles
  4. Homogenate is centrifuged at high speeds to separate different organelles
  5. Supernatant is spun again for a longer time at a higher speed to obtain pellets of less dense organelles.
• Separation of organelles is based on their density 


  Most dense (short time and low speed) --> least dense

  Nuclei, mitochondria, lysosomes, ribosomes