Methods of studying cells

• Microscope


  Instruments that produce a magnified image of an object

  
• Equation for magnification?


  Magnification = image size / real size
• What is resolution?


  the minimum distance apart two objects can be in order for them to appear as separate items.
• what does resolution depend on in microscopes?

  the wavelength of the form of radiation used (visible light VS electron beams).

  
  

  Increasing magnification won't always increase resolution and may present blurry images the more magnified.
• What is the resolution and max magnification of a light microscope

  x2000 mag and 0.2 micrometers (um)
• what wavelength do light microscopes have?

  really long
• How do light microscopes work?

  First light passes through condenser lenses. the visible light then passes through a specimen and then through glass lenses, which magnify the image presenting it on the eye piece lens of which we can see.
• What are some disadvantages to using a light microscope

  - Low resolving power and long wavelength
• what are some advantages of using a light microscope?

  needs hardly any maintenance, not that expensive, can observe living cells
• How do electron microscopes in general work

  they use a beam of electrons inside a vacuum that is first condensed by condenser magnets. the electrons then pass through the object and through the magnetic objective on which an image is displayed on a screen.
• why do electron microscopes need a vacuum


  so particles in the air don't deflect of absorb the electrons
• What are the two electron microscopes?

  TEM (transmission electron microscope) and SEM (scanning electron microscope)

  
• What is the resolution and magnification of the SEM?

  x1,000,000 mag and 20nm
• what his the resolution and magnification of the TEM

  x50,000,000 mag and 0.1nm
• How does the SEM work

  . The image produced is £D directs a beam of electrons onto surface of specimen from above

  
  

  beam passed back and forth across portion of specimen in regular pattern

  
  

  electrons scattered and pattern of these depend on contours of specimen

  
  

  3-D image built by computers analysis of the patterns and secondary electrons produced The image produced is 3D
• How does the TEM work?

  -electron gun produces a beam of electrons- then focused onto specimen by a condenser electromagnet

  
  

  -beam passes through a thin section of specimen- some parts are absorbed by specimen so appear dark and other parts allow to pass through so appear light

  
  

  -an image is produced on a screen and can be photographed to give a photomicrograph. the image produced is 2D
• What are the limitations of an SEM

  - can't observe living specimens

  - complex staining process, may introduce artefacts.

  - specimen must be extremely thin as electrons do not penetrate

  -images may contain artefacts
• what is an artefact?


  Things (like air bubbles) that result from how the specimen has been prepared.
• what are the limitations of the TEM?

  - can't observe living specimens

  - complex staining process, may introduce artefacts.

  - specimen must be extremely thin so electrons are able to pass through

  -images may contain artefacts

  - a higher energy electron beam is required and this could damage the specimen

  -difficulties preparing the specimen may limit resolution may be achieved
• What is cell fractionation?

  The process where cells are broken up and the different organelles they contain are separated out.

  
• What are the 3 conditions that the solution for cell fractionation should take place in?

  cold - reduces enzyme activity that could possibly break down organelles

  
  

  same water potential as the tissue- prevents organelles bursting or shrinking due to somatic gain/loss of water

  
  

  buffered- so pH does not fluctuate. Any change in pH could alter the structure of organelles or how enzymes function
• What are the two stages to cell fractionation?

  Homogenisation and ultracentrifugation
• what is homogenisation?


  Breaking up of cells in a homogeniser (blender). The resultant fluid is know as the homogenate which is then filtered to remove any complete cells and large debris
• What is ultracentrifugation?


  The process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge.

  
• what is a centrifuge?

  a machine that spins tubes of homogenate at very High speeds to create centrifugal force.
• What is the process of ultracentrifugation?

  A) the tube of filtrate is placed in the centrifuge and spun at a slow speed

  B) The heaviest organelles the nuclei are forced to the bottom of the tube where they form a thin sediment

  C) The fluid at the top of the tube is removed leaving just the sediment

  D) the supernatant is transferred to another tube in the centrifuge and spun at a higher speed than before

  E)the next heaviest organelles (mitochondria) are forced to the bottom of the tube

  F)The process continues and the next organelle is lysosomes