light microscopes
use light to form an image
* observe live specimens
* produce colour image
electron microscopes
use electrons to form an image
how does TEM work
- electromagnets to focus a beam of electrons
- This beam of electrons is transmitted through the specimen
- Denser parts of the specimen absorb more electrons so they appear darker on the image
ADV of TEM
- provide high-resolution images
- allows the internal structures to be seen
DISADV of TEM
- only be used with very thin specimens
- cannot be used to observe live specimens
- do not produce a colour image
how does SEM work
- SEMs scan a beam of electrons across the specimen
-This beam bounces off the surface of the specimen and the electrons are detected - forming an image
ADV of SEM
- can be used on thick or 3-D specimens
- They allow 3-D structure of specimens to be observed
DISADV of SEM
- lower resolution images
- They cannot be used to observe live specimens
- They do not produce a colour image
what is the purpose of cell fractionation
to break up cells and separate organelles
what do you do before cf
tissue placed in cold, isotonic buffer solution
why is a homogeniser used
to break the plasma membrane of the cells and releases the organelles into a solution called the homogenate
why is the homogenate filtered
to remove large cellular debris
steps of ultracentrifugation
- filtrate is first spun at a low speed
- largest, heaviest organelles to settle at the bottom of the tube, they form a pellet
- rest of the organelles stay suspended in the solution above the pellet ( the supernatant)
- The supernatant is drained off and placed into another tube, is spun at a higher speed forms another supernatant
- new supernatant is drained off and placed into another tube, is spun at an even higher speed
- process repeated
cf and ultracentrifugation
Break open tissue and filter
In ice cold, isotonic, buffered
Centrifuge and remove cellular debris
Centrifuge at high(er) speed,
heaviest to lightest organelles
Nuclei
Chloroplasts (if carrying out cell fractionation of plant tissue)
Mitochondria
Lysosomes
Endoplasmic reticulum
Ribosomes
