2.1.3 methods of studying cells

• 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
• principles and limitations of optical microscope

  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
• principles and limitations of transmission electron microscope (TEM)

  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
• principles and limitations of scanning electron microscope (SEM)

  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

  line up (scale of) eyepiece graticule with (scale of) stage micrometre

  calibrate eyepiece graticule - use stage micrometre to calculate size of divisions on eyepiece graticule

  take micrometre away and use graticule to measure how many divisions make up the object

  calculate size of object by multiplying number of divisions by size of division

  recalibrate eyepiece graticule at different magnifications
• step 1 -> cell fractionation and ultracentrifugation used to separate cells

  homogenise tissue / use a blender

  disrupts cell membrane, breaking open cells and releasing contents / organelles
• step 2 -> cell fractionation and ultracentrifugation used to separate cells

  place in a 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
• step 3 -> cell fractionation and ultracentrifugation used to separate cells

  filter homogenate

  remove large, unwanted debris eg. whole cells, connective tissue
• step 4 -> cell fractionation and ultracentrifugation used to separate cells

  ultracentrifugation - separates organelles in order of density / mass

  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)