Methods of studying cells

• Cell fractionation


  The process in which different parts and organelles of a cell are separated so that they can be studied in detail
• Differential centrifugation


  Most common method of cell fractionation
• Homogenisation


  1. Cells are first blended in a homogeniser forming the resultant fluid called the homogenate

  2. The homogenate is placed in a centrifuge and spun at a slow speed

  3. The heaviest organelles, the nuclei, are forced to the bottom of the tube where a thin sediment or pellet forms

  4. The supernatant is removed and transferred to another tube and spun at a slightly faster speed, forming a pellet containing the next heaviest organelle, the mitochondria

  5. This process continues so that each time the speed is increased the next heaviest organelles sedimented and separated out
• Homogenisation conditions


  • Performed in a cold, buffered solution of the same water potential as the cells
  • To prevent the organelles from bursting under osmotic pressure
  • To inactivate any enzymes from breaking down organelles
  • So that the pH does not fluctuate
• Light microscope


  Uses a pair of convex glass lenses that can resolve images that are limited by the wavelength of light
• Electron microscope

  • Can distinguish between objects 0.2um apart, compared to 0.7um for light microscopes
  • Two main types: Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)
• Transmission Electron Microscope


  1. A beam of electrons passes through a thin section of a specimen

  2. Areas that absorb the electrons appear darker in the electron micrograph
• Scanning Electron Microscope


  1. A beam of electrons passes across the surface and scatters

  2. The pattern of scattering builds up a 3D image depending on the contours of the specimen
• Limitations of electron microscopes

  • The whole system must be in a vacuum so living specimens cannot be observed
  • A complex staining process is required which may introduce artefacts into the image
  • Specimens have to be very thin, particularly for TEM so that the electrons can pass through
  • SEM has a lower resolving power than TEM, but both have greater resolving power than a light microscope
• Magnification of an image as seen through a microscope can be calculated using the formula: Magnification = size of image/size of real object
• Resolution


  The minimum distance apart that two objects can be distinguished as separate objects in an image. The greater the resolution, the more clear the image will be.