1. Magnification is how much bigger the image is than the specimen (the sample you're looking at). It's calculated using this formula:
Magnification = Length of Image / Length of Specimen
2. Resolution is how detailed the image is. More specifically, it's how well a microscope distinguishes between two points that are close together. If a microscope lens can't separate two objects, then increasing the magnification won't help.
1. Use light.
2. Have a lower resolution than electron microscopes - their maximum resolution is about 0.2 micrometres.
3. The maximum useful magnification of a light microscope is about x1500.
1. Use electrons instead of light to form an image.
2. Have a higher resolution than light microscopes so give a more detailed image - their maximum resolution is about 0.0001 micrometres, 2000 times higher than light microscopes.
3. The maximun useful magnification of an electron microscope is about x1500000.
4. A complete vacuum is required to use them, as electrons can be absorbed by air. This means that living samples can't be used.
Transmission electron microscopes (TEMs)
1. Use electromagnets to focus a beam of electrons, which is then transmitted through the specimen.
2. Denser parts of the specimen absorb more electrons, which makes them look darker on the image you end up with.
3. TEMs are useful because they produce high resolution images.
4. However, they can only be used on thin specimens.
Scanning electron microscopes (SEMs)
1. Scan a beam of electrons across the specimen.
2. Knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image.
3. The images you end up with show the surface of the specimen and they can be 3-D.
4. SEMs are good because they can be used on thick specimens.
5. However, they give lower resolution images than TEMs.
Before fractionation, the tissue must be placed in a cold, isotonic buffered solution. Cold - to reduce enzyme activity as it may break down organelles. Isotonic - prevent organelles from bursting/ shrinking from osmotic gain/loss of water. Buffered- maintain a constant pH.
Homogenisation can be done in several different ways, e.g. by vibrating the cells or by grinding the cells up in a blender. This breaks up the plasma membrane and releases the organelles into solution.
The homogenised cell solution is filtered through a gauze to separate any large cell debris or tissue debris, like connective tissue, from the organelles. The organelles are much smaller than the debris, so they pass through the gauze.
After filtration, you're left with a solution containing a mixture of organelles. To separate a particular organelle from all the others you use ultracentrifugation.
1. The cell fragments are poured into a tube. the tube is put into a centrifuge (a machine that separates material by spinning) and is spun at a low speed. the heaviest organelles, like nuclei, move to the bottom of the tube. They form a thick sediment at the bottom - the pellet. The rest of the organelles stay suspended in the fluid above the sediment - the supernatant.
2. The supernatant is drained off, poured into another tube and spun in the centrifuge at a higher speed. Again, the heaviest organelles, this time the mitochondria, form a pellet at the bottom of the tube. The supernatant containing the rest of the organelles is drained off and spun in the centrifuge at an even higher speed.
3. This process is repeated at higher and higher speeds, until all the organelles are separated out. Each time, the pellet at the bottom of the tube is made of lighter and lighter organelles.