Investigating thestructure of cells

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Microscopy

  • Instruments that magnify the image of an object.
  • Light microscopes have long wavelength of light rays.
  • This means that a light microscope can only distinguish between two objects if they are 0.2 micrometers or more apart.
  • better microscopes used a beam of electrons.
  • Beams of electrons have a shorter wavelength than light.
  • They can distinguish two objects as close as 0.1nm.
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Magnification

  • Material under the microscope is the object.
  • The appearance if the material when viewed under a microscope is reffered to as the image.
  • magnification= size of image/ size of object
  • When calculating this the measurements of the image and the object must be in the same units, e.g. cm.
  • size of object= size of image/ magnification.
  • 1m-1m
  • 1cm= 1/100m
  • 1mm=1/1,000m
  • 1um=1/1,000,000m
  • 1nm=1/1,000,000,000m
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Resolution

  • Resolution or resolving power of a microscope is the minimum distance apart that two objects can be in order for them to appear as seperate objects.
  • Resolving power depends on the wavelength or form of radiation used.
  • Light microscope- 0.2 um
  • Greater resolution means greater clarity.
  • The image produced is clearer and more precise.
  • Increasing the magnification will increase the size of the image, but does not always increase the resolution.
  • Every microscope has a limit of resolution.
  • Up to this point increasing the magnification will reveal more detail but beyond this point  increasing the magnification will not do this.
  • The object, while appearing larger, will just be more blurred.
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Cell Fractionation

  • In order to study the structure and function of various organelles it is necessary to obtain large numbers of isolated organelles.
  • Cell fractionation is the process where cells are broken up and different organelles they contain are seperated out.

Before the process can begin, the tissue is placed in a cold, isotonic, buffered solution.

Cold - to reduce enzyme activity that might break down the organelles.

Isotonic - to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water. It is a solution that has the same water potential as the original tissue.

Buffered - to maintain a constant pH

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Step 1) Homogenation

  • Cells are broken up by a homogeniser (blender)
  • This releases the organelles from the cell
  • The resultant fluid - the homogenate- is filtered.
  • This removes any whole cells or large pieces of debris
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Step 2) Ultracentrifugation

  • The process by which the fragments 0in the homgenate are seperated. This is in a machine called an untracentrifuge. This spins the homogenate at high speeds in order to create a centrifugal force.
  • The process in animals is:
  • 1) The tube of filtrate is placed in the centrifuge and spun at a low speed.
  • 2)The heaviest organelles, the nuclei, are forced to the bottomof the tube where they form a thin sediment of pellet.
  • 3)The supernatant is removed, leaving the sediment of nuclei.
  • 4)The supernatant is transferred to another tube and spun in the ultracentrifuge t a higher speed than before.
  • 5)The next heaviest organelles, the mitochondria, are forced to the bottom of the tube.
  • 6)The process is continued in this way so that, at each increase in speed, the next heaviest organelle is sedimented and seperated out.
  • The order is: - Nuclei, - Mitochondria, - Lysosomes, - Ribosomes.
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