Methods Of Studying Cells

• What are microscopes?


  Instruments that produce a magnified image of an object.
• Magnification - equation?

  Magnification = size of image / size of real object.
• Conversions:

  
  

  km to m

  m to m

  mm to m

  micrometre to m

  nanometre to m

  km to m - x1000

  m to m - x1

  mm to m - /1000

  micrometre - /1000000

  nanometre - /1000000000
• Difference between magnification and resolution?

  Magnification = increasing the size of an image. Up until the limit of resolution, an increase in magnification = an increase in detail.

  
  

  Resolution = minimum distance apart that two objects can be for them to appear as separate items.
• Need to appreciate that...

  ...there was a considerable period of time during which the scientific community distinguished between organelles and artefacts.

  
  

  artefacts = (something in a scientific experiment present due to how expt. was prepared or investigated).
• Why is cell fractionation needed?

  Needed to study the structure and function of the various organelles that make up cells.

  
  

  We need a large number of isolated organelles - can get them via cell fractionation.
• Define cell fractionation.

  The process in which cells are broken up and the different organelles they contain are separated out.
• Describe/Outline the process of Homogenisation.

  
  

  Why a cold, isotonic, buffered solution?

  1. Tissues placed in a cold, isotonic (relative to tissue), buffered solution.

  
  

  2. Cells then broken up by a homogeniser/blender - releases organelles from cell - resultant fluid = homogenate - then filtered to remove any complete cells or large pieces of debris.

  
  

  Cold - to reduce enzyme activity, such as lysozymes, that could break down organelles.

  
  

  Isotonic - same water potential as tissue sample - to preven water moving in or out of the cells by osmosis, causing lysis.

  
  

  Buffered - to prevent changes in pH which could affect/denature enzymes.
• Principles of Optical Microscopes.

  Simple convex glass lenses used in pairs in a compound light microscope - focuses object at a short distance by 1st lens, then magnified by 2nd lens.
• Limitations of Optical Microscopes.

  Light has a relatively long wavelength - low resolution. Can only distinguish between objects 0.2 micrometres apart.
• Principles of Transmission Electron Microscopes.

  1. Electron gun produces e- beam, focused onto specimen by a condenser electromagnet.

  
  

  2. Beam passes through a thin section of the specimen from below. Parts absorb e- and appear dark; others let e- pass through and appear bright - produces image on screen - photomicrograph.
• Limitations of Transmission Electron Microscopes.

  1. Can't work on living specimens - needs to be in a vacuum.

  
  

  2. Complex staining process.

  
  

  3. Image not in colour.

  
  

  4. Extremely thin specimens only.

  
  

  5. May contain artefacts, which appear on the finished photomicrograph.

  
  

  But, can get over the flat 2D image, by taking a series of sections through a specimen - can build up a 3D image from these photomicrographs.

  
  

  Max resolution is 0.1 nm but can't always be achieved because:

  
  

  1. Difficulties in specimen preparation.

  2. Higher energy e- beam required, which may destroy the specimen.
• Principles of Scanning Electron Microscopes.

  1. Beam of e- directed onto surface of specimen - passed back and forth across specimen.

  
  

  2. e- scattered by specimen - scattering pattern analysis allows us to get a 3D image.
• Limitations of Scanning Electron Microscopes.

  1. Same as with TEMs, but samples do not need to be thin.

  
  

  2. Lower resolution than TEMs , at 20 nm.
• Describe how you would make a temporary mount (of plant tissue) (4).

  M1 Add drop of water to glass slide.

  M2 Obtain thin section of (plant tissue) and place on slide.

  M3 Stain with x - iodine in KI solution if testing for starch grains etc.

  M4 Lower cover slip using mounted needle.

  
  

  Ensure method avoids trapping air bubbles!
• Graticule? Why needed?

  Glass disc with an etched scale placed in the eyepiece of a microscope.

  
  

  Needed to measure size of objects under objective lens, need to calibrate the eyepiece graticule - each objective lens will magnify to a different degree.
• Describe how to calibrate the eyepiece graticule.

  1. Use a stage micrometer = special microscope slide with an etched scale - line up scales on graticule and micrometer.

  
  

  2. Once lined up, can calculate length of divisions on eyepiece graticule.

  
  

  x40 mag gives 25microm per graticule unit

  
  

  therefore, x400 mag gives 25/10microm per graticule unit.

  
  

  NB = Only need to calibrate once, providing the same objective lens is used.
• How are cells specialised in complex multicellular organisms?

  Cells initially all identical in an embryo but as they develop, the switching on/off of certain genes takes place, leading to changes in the organelle numbers and shapes of cells.
• Define tissue.

  
  

  Give an example.

  Collection of similar cells that are aggregated together and work together to perform a specific function.

  
  

  Example = epithelial tissue - consists of sheets of cells, lining the surfaces of organs, often having a protective or secretory function.
• Define organ.

  
  

  Comment on the difference between capillaries, veins and arteries, relative to the term organ.

  Combination of aggregated tissues that are co-ordinated together to perform a variety of functions, one of which is the predominant major function.

  
  

  While capillaries, veins and arteries all have the same major function, i.e.e carrying blood, capillaries are not organs, unlike veins and arteries as they are made up of only one tissue - epithelium.
• Define organ system.

  
  

  Give examples.

  Organs working together as a single unit - systems may be grouped together to perform particular functions more efficiently.

  
  

  Digestive, Respiratory, Circulatory systems etc.
• Define eukaryotic cell.

  Larger cells with a true nucleus bounded by nuclear membrane/nuclear envelope.
• Define prokaryotic cell.


  Smaller, have no true nucleus or nuclear envelope.