microscopes/cell fractionation

• What is magnification?

  • The magnification of an object is how many times bigger the image is when compared to the object
• What is resolution?

  • The resolution of a microscope is the minimum distance apart that two objects can be in order for them to appear as separate items. 
• How does an optical (light) microscope work?

  • They use light to form an image. 
  • They have a maximum resolution of about 0.2 micrometres (µm).
  • Low resolution as wavelength of light is too long. 
  • Can see ribosomes, the endoplasmic reticulum and lysosomes, mitochondria and nucleus. 
• How does an electron microscope work?

  • Use electrons to form an image. 
  • Have a higher resolution than optical microscopes, so has more detailed image (can see more organelles). 
  • Maximum resolution of about 0.0002 micrometres (µm).
  • Electron microscopes produce black and white images
• How does a TEM work?

  TEMs use electromagnets to send a beam of electrons through a specimen. 

  Denser parts of the specimen absorb more electrons, which makes them look darker on the image you end up with. 
• What are the advantages of using a TEM?

  • Gives high-resolution images, this allows the internal structures within cells/organelles to be seen
  • Higher magnification
• What are the disadvantages of using a TEM?

  • Can only be used with very thin specimens or thin sections of the object being observed
  • Cannot be used to observe live specimens (vacuum inside TEM meaning all the water must be removed from specimen, unlike optical microscopes that can be used to observe live specimens)
  • Only black and white images
• How does an SEM work?

  • SEMs scan a beam of electrons across the specimen. 
  • The images show the surface of the specimen and they can be 3D.
• What are the advantages of using an SEM?

  
  

  They can be used on thicker specimens

  They allow the external of specimens to be observed

  Produces 3D images
• What are the disadvantages of using an SEM?

  • They give lower resolution images (less detail) than TEMs
  • They cannot be used to observe live specimens (unlike optical microscopes that can be used to observe live specimens)
• Similarities between SEM and TEM

  • Both use electrons
  • Both have higher resolutions/magnification than optical microscopes
• Differences between SEM and TEM

  • SEM can produce 3D images whilst TEM cannot
  • TEM has higher maximum resolution than SEM
  • SEM can be used on thicker specimens than TEM
• What is cell fractionation?

  Cell fractionation is the process where cells are broken up and the different organelles they contain are separated out. 
• Why is the tissue placed in an cold, isotonic, and buffered solution before fractionation?

  • 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  
  • Buffered - so that the pH docs not fluctuate. Any change in pH could denature enzymes/proteins
• What is homogenistaion?

  • The tissue-containing solution is then homogenised using a homogeniser 
  • This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate
• What is filtration?

  • The homogenate is then filtered through a gauze to separate out any large cell debris or tissue debris 
  • The organelles are all much smaller than the debris and are not filtered out (they pass through the gauze)

  This leaves a solution (known as the filtrate) that contains a mixture of organelles
• What is ultracentrifugation?

  Ultracentrifugation is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge. 
• What are the heaviest organelles in order?

  • nuclei, chloroplast, mitochondria, lysosomes, endoplasmic reticulum, ribosomes 
• 1st step of ultracentrifugation

  • The filtrate is first spun at a low speed. This causes the largest, heaviest organelles (such as the nuclei) to settle at the bottom of the tube, where they form a thick sediment known as a pellet. The rest of the organelles stay suspended in the solution above the pellet. (This solution is known as the supernatant)
• 2nd step of ultracentrifugation

  The supernatant is drained off and placed into another tube, which is spun at a higher speed. Once again, this causes the heavier organelles (such as the mitochondria) to settle at the bottom of the tube, forming a new pellet and leaving a new supernatant
• 3rd step of ultracentrifugation

  • The new supernatant is drained off and placed into another tube, which is spun at an even higher speed. This process is repeated at increasing speeds until all the different types of organelle present are separated out (or just until the desired organelle is separated out). Each new pellet formed contains a lighter organelle than the previous pellet