Investigating cells

microscopy
1. Lenses work more effectively if they are in a compound light microscope
2. Light waves a have a relatively long wavelength; therefore, they can only distinguish between objects that are at least 0.2 micrometers apart
3. Beams of electrons have shorter wavelengths and are therefore able to distinguish between objects as close as 0.1 nm apart
Magnification
1. When viewed under a microscope, the material seen in called an image
2. Magnification tells you how many times bigger the image is in relation to the actual size of the object
3. Magnification = size of image/size of object
Resolution
1. The resolving power of a microscope is the minimum distance two objects can be apart in order for them to appear as separate items
2. The greater the resolution, the greater the clarity of the image that is produced
Cell fractionation
1. Cell fractionation is the process where cells are broken up and the different organelles they contain are separated out
2. Before fractionation begins, the cells are put in a solution that is:
  1. Cold
    1. to reduce enzyme activity that might break down the organelles.
  2. Isotonic
    1. to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water. An isotonic solution is one that has the same water potential as the original tissue.
  3. Buffered
    1. to maintain a constant pH
Homogenation
1. Cells are broken up by a homogeniser that releases the organelles
  1. The fluid is called a homogenate
    1. It is then filtered to remove complete cells and large pieces of debris
Ultracentrifugation
1. Ultracentrifugation is the process by which the homogenate is separated in a machine called a centrifuge
  1. This spins tubes of the homogenate, creating a centrifugal force that makes the mixture separate
    1. The tube of filtrate is placed in the ultracentrifuge and spun at a slow speed
      1. The heaviest organelles such as the nucleus are forced to the bottom where they form a thin sediment
        The fluid at the top, called the supernatant is removed, leaving just the sediment of nuclei at the bottom
        The supernatant is then put in another tube where it is spun at an even higher speed than before
        The next heaviest organelles (mitochondria) are forced to the bottom and the process continues until all the organelles are separated
electron microscope
1. Electrons have a shorter wavelength than light and so they have a greater resolving power
2. As electrons are negatively charged, the beam can be focused using an electromagnet
3. Because electrons are absorbed by molecules in the air, a near vacuum must be created within the chamber of an electron microscope for it to work effectively
4. Transmission electron microscope
  1. The TEM consists of a gun that fires electrons which are focused onto the specimen by an electromagnet
    1. Some of the electrons are absorbed by the specimen and appear dark on the image; other parts allow the electrons through and so appear light. This produces an image of the specimen
      1. The image that appears on screen is called a photomicrograph
        Because the process takes place in a vacuum, living specimens cannot be observed
        A complex staining process is required and even then the image is only in B&W
        The specimen must be extremely thin
        Artefacts (structure not present in the organism when it was alive) may appear on the image, these appear as a result of the way the specimen is prepared
5. Scanning electron microscope
  1. All the limitations of the TEM apply to the SEM but the specimen does not have to be extremely thin as the electrons do not penetrate
    1. The beam of electrons is directed over the surface of the specimen in a regular pattern
      1. The electrons bounce on the contours of the specimen and are scattered
        The scattering of the electrons can be analysed and from this an image can be produced using a computer
        The SEM has a lower resolving power than the TEM (20nm) but is still ten times better than a light microscope

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Investigating cells

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