3.1 & 3.2 Methods of studying Cells and The Electron Microscope

3.1 & 3.2 Methods of studying Cells and The Electron Microscope

1. Optical Microscopes

   • Use light to form an image
   • Maximum resolution of about 0.2micrometres
     • So you can't use an optical microscope to view organelles smaller than 0.2 micrometres. This includes ribosomes, ER and lysosomes. You may see mitochondria, but not in peprfect detail.
   • Maximum useful magnification of about x1500
   • Using a slide to view specimens
     • 1) add a small drop of water onto the slide then use tweezers to place a thin section of your specimen on top of the water drop
     • 2) Add a drop of stain to highlight objects in the cell (e.g Iodene is used for starch)
     • 3) Finally, add a cover slip. Careful for air bubbles!

2. Transmission Electron Microscope (TEM)

   • Use electromagnets to focus a beam on electrons which is then transmitted through the specimen
     • Electron beam has a very short wavelength
       • High resolving power of 0.1nm
   • Denser parts of specimen absorb more electrons = darker on image
   • High resolution images = see internal structure of organelles such as chloroplasts
   • Only can be used on thin specimens
   • Limitations
     • Whole system is in a vacumn = specimens must be dead
     • Specimen has to undergo a complex staining process = can be damaged, not true colour.
     • Specimen has to be extremely thin
     • Image may contain artefacts ( things resulting from the way the specimen is prepared) = foreign objects on end photomicrograph

3. Scanning Electron Microscope (SEM)

   • Scan a beam of electrons across the specimen
     • knocks off electrons from the specimen which are gathered in a cathode ray tube to form an image
   • Images show surface of specimen and so they can be 3D
   • They have lower resolution than a TEM
     • 20nm
   • The Limitations of a SEM are very similar to a TEM, however the specimen doesn't need to be extremely thin as electrons do not penetrate it

4. Magnification is Size

   • Magnification = Image size/Actual size
   • Magnification is how much bigger the image is than the specimen

5. Resolution is detail

   • Resolution is how detailed the image is, and how well a microscope distinguishes between two points that are close together

6. Cell Fractionation and Ultracentrifugation

   • If you want to look at some organelles under an electron microscope, you'd first need to separate them from the rest of the cell = cell fractionation.
     • 1) Homogenisation - Breaking up the cells
       • Cells are broken up by a homogeniser (blender). This breaks up the plasma membrane and releases the organelles into solution. The solution is called homogenate.
         • Solution has to be ice-cold = reduce activity of enzymes that break down organelles
         • Solution has to be Isotonic = same concentration of chemicals as the cells being broken down/same water potential as tissue to prevent organelles bursting/shrinking.
         • A buffer solution should also be added to homogenate to maintain pH as a change in pH could alter the structure of the organelles
     • 2) Filtration - Getting rid of the big bits
       • The homogenate is filtered through a gauze to seperate any large cell debris or tissue debris from the organelles
         • The organelles are much smaller than the debris so they pass through the gauze
     • 3) Ultracentrifugation - Separating the organelles
       • Separated in a machine called the centrifuge
       • The tube of filtrate is placed in the centrifuge and spun at low speed
         • The heaviest organelles, the nuclei, are forced to the bottom of the tube and form thing sediment
           • Fluid at the top of the tube (supernatant) is removed, leaving just the sediment of nuclei
             • The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before
               • Next heaviest organelles (e.g mitochondria) forced to the bottom of the tube
                 • The process is continued in this way so with each increase in speed the next heaviest organelle is sedimented and separated