analysis of cell components

• magnification is how much bigger the image is than the specimen (sample looking at)
• magnification = size of image / size of real object
• mm - micrometer = x1000

  micrometer - nm = x1000
• 1mm = 1mm
• 1 micrometer = 0.001 mm
• 1 nm = 0.000001 mm
• resolution = how detailed the image is.

  how well a microscope distinguishes between 2 points that are close together

  if a microscope lens cannot separate 2 objects, increasing the magnification will not help
• optical (light) microscope

  • use light to form an image
  • max resolution of about 0.2 micrometres
  • can't use an optical microscope to view organelles smaller than 0.2 microm - includes ribosomes, endoplasmic reticulum and lysosomes
  • may be able to make out mitochondria - not in perfect detail
  • can see the nucleus
  • max useful magnification of an optical microscope is about x150
• electron microscopes

  • use electrons to form an image
  • higher resolution than optical microscopes - give a more detailed image - can be used to look at more organelles
  • max resolution of about 0.0002 microm (1000x higher than optical microscope)
  • max useful magnification is about 1500000x
  • produce black and white images - often coloured by a computer
• transmission electron microscope

  • use electromagnets to focus a beam of electrons which is then transmitted through the specimen
  • denser parts of the specimen absorb more electrons which makes them look darker on the image
  • high resolution images - see internal structure of organelles like chloroplasts
  • have to view the specimen in a vacuum - can't look at living organisms
  • only used on thin specimens
• scanning electron microscope:

  • scan a beam of electrons across the specimen
  • knocks off electrons from the specimen - gathered in a cathode ray tube to form an image
  • images you end up with show the surface of the specimen and can be 3D
  • can be used on think specimens
  • lower resolution images than TEMS
• Preparing microscope slides


  1. Put specimen on microscope slide

  2. Use a temporary mount (wet mount) to suspend specimen in a drop of liquid

  3. Pipette a small drop of water onto the centre of the slide

  4. Use tweezers to place a thin section of the specimen on top of the water drop

  5. Add a drop of stain
• Temporary mount (wet mount)


  Where the specimen is suspended in a drop of liquid (e.g. water, oil) on the slide
• Specimen


  • Needs to let light through to be able to see it clearly under the microscope
  • If have a thick specimen, need to take a thin slice for use on slide
• Stain


  Used to highlight objects in a cell
• eosin is used to make the cytoplasm show up
• iodine in potasium iodide solution is used to stain starch grains in plant cells
• finally add the cover slip (square of clear glass or plastic that protects the specimen):

  • to do so stand the slip upright on the slide next to the water droplet
  • then carefully tilt and lower it so it covers the specimen
  • try not to get any air bubbles under it - they will obstruct the view of the specimen
• microscope artefacts - artefacts are things you can see down the microscope that are not part of the cell or specimen you are looking at - can be anything from bits of dust, air bubbles and finger prints, to inaccuracies caused by squashing or staining your sample
• artefacts are usually made during the preparation of your specimen and shouldn't really be there at all
• artefacts are especially common in electron micrographs bc specimens need a lot of preparation before you can view them under an electron microscope
• homogenisation - breaking up the cell

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

  • homogenised cell solution is filtered through a gauze to separate any large cells or tissue debris like connective tissue, from the organelles
  • the organelles are much smaller than the debris so they pass through the gauze
• ultracentrifugation:

  • use to separate solution containing mixture of organelles
  • cell fragments are poured into a tube - tube put into a centrifuge (machine that separates materials by spinning) and is spun at a low speed
  • heaviest organelles like nuclei get flung to the bottom of the tube by the centrifuge
  • form a thick sediment at the bottom - the pellet
  • rest of the organelles stay suspended in the fluid above the sediment - supernatant
• • supernatant is drained off, poured into another tube and spun in the centrifuge at a higher speed
  • again heaviest organelles form pellet at bottom of the tube
  • supernatant containing rest of the organelles is drained off and spun in centrifuge at even higher speed
• • process repeated at higher and higher speeds until all the organelles are separated out
  • each time pellet at the bottom of the tube is made up of lighter and lighter organelles
• organelles are separated in order of mass (heaviest to lightest)

  usually:

  • nuclei
  • mitochondria
  • lysosomes
  • endoplasmic reticulum
  • ribosomes
  • in plant cells chloroplasts come after nuclei but before mitochondria