2.1.3 Methods of studying cells

• Describe the difference between magnification and resolution

  ● Magnification = number of times greater image is than size of the real (actual) object

  ○ Magnification = size of image / size of real object

  ● Resolution = minimum distance apart 2 objects can be to be distinguished as separate objects
• Describe the principles and limitations of optical microscopes

  • Light focused using glass lenses
  • Light passes through specimen, different structures absorb different amounts & wavelengths
  • Generates a 2D image of a cross-section
  • Low resolution due to long wavelength of light
  • Can't see internal structure of organelles or ribosomes
  • Specimen = thin
  • Low magnification (x 1500)
  • Can view living organisms
  • Simple preparation
  • Can show colour
• Describe the principles and limitations of transmission electron microscopes

  Electrons focused using electromagnets

  Electrons pass through specimen, denser parts absorb more and appear darker

  Generates a 2D image of a cross-section

  Very high resolution due to short wavelength of electrons

  Can see internal structures of organelles and ribosomes

  Specimen = very thin

  High magnification (x 1,000,000)

  Can only view dead / dehydrated specimens as uses a vacuum

  Complex preparation so artefacts often present

  Does not show colour
• Describe the principles and limitations of scanning electron microscopes

  • Electrons focused using electromagnets
  • Electrons deflected/bounce off specimen surface
  • Generates a 3D image of surface
  • High resolution due to short wavelength of electrons
  • Can't see internal structures
  • Specimen does not need to be thin
  • High magnification (x 1,000,000)
  • Can only view dead / dehydrated specimens as uses a vacuum
  • Complex preparation so artefacts often present
  • Does not show colour
• Describe how the size of an object viewed with an optical microscope can be measured

  1. Line up (scale of) eyepiece graticule with (scale of) stage micrometre
  2. Calibrate eyepiece graticule - use stage micrometre to calculate size of divisions on eyepiece graticule
  3. Take micrometre away and use graticule to measure how many divisions make up the object
  4. Calculate size of object by multiplying number of divisions by size of division
  5. Recalibrate eyepiece graticule at different magnifications
• Describe the process of cell fractionation and ultracentrifugation

  1. Homogenise tissue / use a blender

  2. Place in a cold, isotonic, buffered solution

  3. Filter homogenate

  4. Ultracentrifugation - separates organelles in order of density / mass
• What is the need to homogenise tissue / use a blender

  • Disrupts cell membrane, breaking open cells and releasing contents / organelles
• What is the need to place homogenate in cold, isotonic, buffered solution

  • Cold to reduce enzyme activity → so organelles not broken down / damaged
  • Isotonic so water doesn't move in or out of organelles by osmosis → so they don't burst
  • Buffered to keep pH constant → so enzymes don't denature
• What is the process of ultracentrifugation?

  • Centrifuge homogenate in a tube at a high speed
  • Remove pellet of heaviest organelle and respin supernatant at a higher speed
  • Repeat at increasing speeds until separated out, each time pellet made of lighter organelles (nuclei → chloroplasts / mitochondria → lysosomes → ER → ribosomes)