Methods of studying cells

• Microscopes can be used to analyze cell components and observe organelles
• Two important scientific terms in microscopy are:

  • Magnification: tells you how many times bigger the image produced by the microscope is than the real-life object you are viewing
  • Resolution: the ability to distinguish between objects that are close together
• There are two main types of microscopes:

  • Optical (light) microscopes
  • Electron microscopes
• Optical microscopes use light to form an image, limiting their resolution to around 0.2 micrometers (µm) or 200 nm
• Optical microscopes have a maximum magnification of about ×1500
• Electron microscopes have a maximum resolution of around 0.0002 µm or 0.2 nm, about 1000 times greater than that of optical microscopes
• Advantages of Transmission Electron Microscopes (TEMs):

  • Give high-resolution images
  • Allow the internal structures within cells to be seen
• Disadvantages of Transmission Electron Microscopes (TEMs):

  • Can only be used with very thin specimens
  • Cannot be used to observe live specimens
  • Lengthy treatment required to prepare specimens can introduce artefacts
  • Do not produce a color image
• Advantages of Scanning Electron Microscopes (SEMs):

  • Can be used on thick or 3-D specimens
  • Allow the external, 3-D structure of specimens to be observed
• Resolution is the ability to distinguish between two separate points
• If two separate points cannot be resolved, they will be observed as one point
• The resolution of a light microscope is limited by the wavelength of light
• As light passes through the specimen in a light microscope, it will be diffracted
• The longer the wavelength of light, the more it is diffracted and the more that this diffraction will overlap as the points get closer together
• Electron microscopes have a much higher resolution and magnification than a light microscope as electrons have a much smaller wavelength than visible light
• What are the three stages of cell fractionation

  Homogenisation

  filtration

  ultrafiltration
• homogenisation

  breaks up cells in a tissue sample.

  place sample in an ice-cold, buffered and isotonic solution

  cold - to prevent enzyme activity (slow metabolic reactions)

  buffered - maintains pH, preventing enzymes from denaturing

  isotonic - prevent organelles from being damaged by osmosis
• filtration

  removed large cell debris/ unbroken cells

  • the solution (homogenate) is passed through a filter, any large debris and unbroken cells is removed - (filtrate)
• ultrafiltration 


  the filtrate is placed into a centrifuge

  • spun at low speeds - causing the heavier organelles to fall and form the pellet and the lighter organelles remain at the top to form a supernatant
  • this is repeated, increasing the speeds each time.

  nuclei > chloroplast > mitochondria > lysosomes > ER > ribosomes [n.c.m.l.e.r]
• When looking at a prepared sample under a microscope you can sometimes see things that aren’t actually part of the specimen. These are known as artefacts. Artefacts can be dust, air bubbles or fingerprints.
• how do you decrease the occurrence of artefacts?

  repeat and compare between samples. if a particular object is present in one specimen but not present in another the object is most likely an artefact.
• What are the principles of using a transmission electron microscope ?

  TEMs use electromagnets to focus beam of electrons

  This beam of electrons is transmitted through the specimen

  The darker parts of the specimen absorb more electrons
• What are the principles of using a scanning electron microscope ?

  -SEMs scan a beam of electrons across the specimen

  -This beam bounces off the surface of the specimen and the electrons are detected, forming an image (therefore can produce three-dimensional images)