microscopes and analysis of cells

• light microscope -

  • long wavelength - relatively low resolution (0.2 micrometers) and low magnification
• transmission electron microscope -

  • 2D image
  • dead specimen only
  • high resolution and high magnification
  • specimen must be in a vacuum
• scanning electron microscope -

  • 3d image
  • dead specimen only
  • high resolution and magnification
  • specimen must be in a vacuum
• using a graticule

  • line up graticule with stage micrometer
  • count no. of divisions in a known length, provided by stage micrometer
  • divide known length by no. of divisions to find length of one division
• ice cold - to reduce kinetic energy of enzymes to prevent them from breaking the cells down
• buffered - same pH inside and outside the cell to prevent break down of proteins in the cell
• isotonic - to make sure water doesn’t enter cell via osmosis to prevent the cell from bursting
• cell fractionation

  
  

  homogenisation

  • place tissue in ice cold, buffered, isotonic solution
  • cells are then broken up by a homogeniser
  • resultant fluid (homogenate) is filtered to remove large pieces of debris
• filtration

  • homogenate is passed through a gauze filter, removing large debris
• ultracentrifugation

  • first spins at lowest speed
  • after some spins , pellet is formed, most dense organelle forms pellet first (usually the nucleus)
  • remove pellet and centrifuge again at a higher speed
  • pellet of second most dense organelle will form
  • repeat until desired pellet is formed