2.1.3 - Analysis of Cell Components

• Magnification is the number of times larger an image is than the specimen being examined
• magnification = (image size / actual size) x 100
• when calculating magnification, all units must be in the same format e.g. all in millimetres
• 1mm = 1000um = 1,000,000nm
• 1nm = 0.001um = 0.000001mm
• resolution is how detailed an image is
• resolution is defined as how well a microscope distinguishes between two points that are close together
• there are two types of microscope:

  • light microscope
  • electron microscope
• light microscopes:

  • use light to form an image
  • maximum resolution of 0.2um
  • maximum magnification of x1500
• electron microscopes:

  • use electrons to form an image
  • higher resolution that light microscopes with a maximum resolution of 0.0002um
  • maximum magnification of x1,500,000
• there are two types of electron microscope:

  • transmission electron microscope (TEM)
  • scanning electron microscope (SEM)
• transmission electron microscopes:

  • use electromagnets to focus a beam of electrons which is transmitted through the specimen
  • denser parts absorb more electrons so appear darker on the final image
  • have a higher resolution than SEMs
  • specimen must be viewed in a vacuum
  • specimen must be thin
• scanning electron microscope:

  • scan a beam of electrons across the specimen which knocks off electrons from the specimen which are gathered in a cathode ray tube, forming an image
  • image shows the surface of the specimen and can be 3D
  • can be used on thick specimens
  • have a lower resolution than TEMs
• preparing a microscope slide (temporary mount):

  1. pipette a small drop of water onto the middle of the slide
  2. use tweezers to place a thin section of specimen on top of the water drop
  3. add a drop of stain
  4. add the cover slip at an angle trying not to get any air bubbles
• stains are used on microscope slides to highlight objects in a cell
• different stains highlight different objects in cells:

  • eosin highlights the cytoplasm
  • iodine in potassium iodide solution highlights starch grains in plant cells
• microscope artefacts are objects that are seen down the microscope but aren’t part of the specimen. they are usually made during preparation of the specimen
• examples of artefacts:

  • dust
  • air bubbles
  • fingerprints
  • inaccuracies caused by squashing and staining a sample
• artefacts are especially common in electron micrographs due to the high requirement of specimen preparation
• cell fractionation is the process of separating a cell into its different parts
• there are three steps to cell fractionation:

  1. homogenisation
  2. filtration
  3. ultracentrifugation
• homogenisation (breaking up of cells) can be done by vibrating the cells or grinding them up in a blender, this breaks down the cell surface membrane, releasing the organelles into solution
• filtration (removing the large debris) is done by passing the homogenised cell solution through a gauze
• ultracentrifugation (separating the organelles) is done by:

  • pouring the cell solution into a tube and placing it into a centrifuge
  • centrifuge at a low speed to separate the heaviest organelles i.e. nuclei to the bottom of the tube
  • the sediment at the bottom is called the pellet and the organelles still suspended in fluid is the supernatant
  • the supernatant is drained off into another tube and centrifuged again at a higher speed
  • the supernatant is then drained off from the pellet into another tube and the process is repeated at higher speeds each time
• order of organelle separation in ultracentrifugation:

  1. nuclei
  2. chloroplasts
  3. mitochondria
  4. lysosomes
  5. endoplasmic reticulum
  6. ribosomes
• there are three conditions required for cell fractionation:

  • cold - to reduce enzyme activity
  • isotonic - ensure there is the same concentration of chemicals as cells being broken down to reduce damage to cells by osmosis and lysis
  • buffered - to maintain pH