methods of studying cells

• optical light microscopes use light to form an image

  they have a max resolution of about 0.2 micro metres

  max useful magnification is x1500
• light image

  longer wavelength

  lower magnification
• electron microscope

  use electrons to form an image

  higher resolution

  max useful mag x1,500,00

  black and white images
• electron microscopes use electrons which have a shorter wavelength
• calibrating the eyepiece graticule

  apparatus:

  mounted needle

  microscope

  stain

  white tile

  glass slide

  cover slip

  stage micrometer

  eye peice graticule

  slide
• calibrating the eye piece graticule

  1. prepare a temporary mount of a cell
  2. unscrew eye piece
  3. identify the black needle in the eye piece
  4. insert EPG into the eye piece gently and adjust it so it rests on top of the needle, use a mounted needle to help
  5. screw eye piece lens back on
  6. set lens at x4
  7. focus the stage micrometer image
  8. twist the eyepiece lens to line up with the 0 of the EPG with the beginning of the stage micrometer
  9. count how many EPG units there are across the 1mm stage micrometer scale and record
• light has a longer wavelength so lower resolution
• electrons have a shorter wavelength so have a higher resolution
• electrons can only operate in a vaccum
• TRANSMISSION ELECTRON MICROSCOPE

  • electron gun that produces beam of electrons that focused onto the specimen by a condenser electromagnet
  • the beam passes through a thin section of the specimen
  • parts of the specimen absorbs electrons so appear dark
  • other parts allow the electrons through so appear bright
  • an image produce called photomicrograph
• resolving power of TEM 0.1nm
• the TEM can have disadvantages

  • must be in a vacuum so living specimens can’t be observed
  • complex staining process
  • not in colour
  • must be extremely thin
  • may contain artefacts not part of og
• SCANNING ELECTRON MICROSCOPE

  • the specimens dont need to be extremely thin
  • the SEM directs a beam of electrons onto the surface of the specimen from above rather than penetrating it
  • the beam is then passed back and forth
  • the electrons are scattered by the specimen and the pattern depends on the contours of the specimen surface
  • 3-D image by the computer analysis of the pattern of scattered electrons
  • lower resolving power than TEM 20nm
• The TEM has a 2-D image micrograph

  the denser regions are the darker
• TEM ADVANTAGE

  • resolving power 0.1nm
  • dark/bright regions
• SEM ADVANTAGE

  • specimens don’t need to be extremely thin as TEM
  • 10x better than light
  • 3-D image
  • can be used on thick specimens
• /SEM DISADVANTAGES

  • must be in a vacuum
  • living specimens can’t be observed
  • complex staining process
  • not in colour
  • may contain artefacts
  • a lower resolving power than TEM at 20nm
• artefacts are things that appear down a microscope that aren’t part of the specimen

  • dust
  • air bubbles
  • errors during slide prep
• resolution is the minimum distance apart that the two objects can be in order for them to appear as separate objects
• To look at the structure of a specific organelle

  • homogenisation
  • filtration
  • ultracentrifugation
• step 1 HOMOGENISATION

  • this breaks up the plasma membrane releasing organelles
  • the fluid called homogenate
  • this can be done by vibrating the cells often using sound waves or grinding the tissue in a blender
  • ice cold, isotonic, buffered
• Ice cold:

  reduce enzyme activity

  to avoid the break down of organelles
• Isotonic (balanced):

  solution is the same water potential as the organelles

  so avoids water moving in or out by osmosis which could burst or distort their shape
• buffered:

  maintains a normal pH of the organelles so

  proteins are not denatured
• FILTRATION:

  • using a filter funnel and filter paper to filter and remove unbroken cells or large pieces of cell debris
• Ultracentrifugation involves spinning the filtrate at high speeds to separate different components based on their size and density.
• heaviest to lightest

  • nuclei
  • chloroplast
  • mitochondria
  • lysosome
  • er
  • ribosome
• heaviest to lightest

  • nuclei
  • chloroplast
  • mitochondria
  • lysosome
  • er
  • ribosome
• cell contents at bottom of the tube

  • pellet
• the larger most dense cells must be spun at a lower speed because less gravitational force is needed to force them down into the pellet
• the liquid contents not in the pellet is the supernatent
• the liquid is seperated from the pellet by the centrifugal forces/ being spun
• the liquid is seperated by a pippete
• the two parameters used when separating organelles during ultracentrifugation is mass and density of cells
• preparing a microscope slide

  1. prepare slide by adding a water droplet on a clean slide temporary mount: specimen suspended in a drop of liquid on a microscope slide
• preparing a microscope slide

  2. add the specimen you want to view on top of the water drop

  the specimen has to be thing for light to pass through
• preparing a microscope

  step 3. add a stain on top of specimen which allows to see organelles better, as a stain clings to organelles to highlights them