Microscopes & Ultracentrifugation

• Describe how light microscopes work: advantages & disadvantages

  1. light rays are shone through the specimen to make it visible
  2. a stain is used to make the object clearer
  3. reflected light is transmitted to observer

  advantages:

  • cheap
  • easy to use
  • small

  disadvantages:

  • low magnification
  • low resolution
  • produces flat images
• Describe how a TEM work: advantages & disadvantages

  1. shines a beam of electrons through thin specimen
  2. dense structures absorb more electrons
  3. cells are stained

  advantages:

  • high magnification
  • high resolution
  • high quality image

  disadvantages:

  • can only examine dead cells
  • sample has to be thin - prep can take a while
  • expensive
  • need to be highly skilled to use
  • very large
• Describe how the SEM works

  1. shines beam of electrons onto surface of specimen
  2. electrons are reflected to the detector
  3. produces a 3D image of the surface of the tissue
  4. specimen is coated in gold in order to reflect electrons

  advantages:

  • high magnification
  • high resolution
  • high quality image
  • 3D image - shows contours

  disadvantages:

  • can only examine dead cells
  • sample prep can take a while
  • expensive
  • higher risk of radiation
  • need to be highly skilled to use
• Define magnification and resolution

  magnification -> the factor by which the image is larger than the actual specimen

  resolution -> the smallest distance between 2 structures at which they can be distinguished from each other
• State the equation for the actual size of a structure

  actual size = image size / magnification
• Outline the processes cell fractionation and ultracentrifugation

  1. cut and homogenize the tissue to break open the cells and release organelles
  2. filter the homogenate to remove debris
  3. spin homogenate in centrifuge
  4. the most dense organelle will form a pellet
  5. filter out the supernatant and spin again at a higher speed
  6. continue until you separate the desired organelle
• state the order of sedimentation of organelles: most -> least

  1. nucleus
  2. mitochondria
  3. lysosomes
  4. RER
  5. plasma membrane
  6. SER
  7. ribosomes
• Explain why fractionated cells are kept in a cold, buffered, isotonic solution

  cold: slow action of enzymes

  buffered: maintain pH

  isotonic: prevent lysis / shrinking of organelles