DNA and cells - MICROSCOPY Flashcards
What is the relationship between magnification, image size and actual size?
Magnification = Image size/Actual size
What is the definition of resolution?
The minimum distance apart two objects can be in order for them to appear as separate items.
Does increasing the magnification affect resolution?
Not always - every microscope has a limit of resolution; after this point, the object may be larger, but more blurred.
What is cell fractionation?
The process where cells are broken up and the different organelles they contain are separated.
What needs to happen before cell fractionation can occur?
The tissue must be placed in a solution that is:
- cold
- buffered
- of the same water potential as the tissue
Before cell fractionation, why does the solution need to be cold?
To reduce enzyme activity that might break down the organelles
Before cell fractionation, why does the solution need to be buffered?
- To maintain cell pH
- A change in pH might alter the structure of the organelles or affect the enzymes
Before cell fractionation, why does the solution need to be isotonic (of the same water potential) as the tissue?
To prevent organelles bursting or shrinking due to osmotic gain or loss of water
What is the first stage of cell fractionation?
HOMOGENATION
- Cells are broken up by a homogeniser
- Releasing the organelles in the cell
- The homogenate (resultant fluid) is then filtered to remove any complete cells and debris
What is the second stage of cell fractionation?
ULTRACENTRIFUGATION
- The tube of filtrate is spun at low speeds in a centrifuge
- The heaviest organelles form a sediment at the bottom
- The supernatant (fluid at the top) is removed
- The supernatant is put in another tube and spun at higher speed in a centrifuge
- The mitochondria are forced to the bottom
- The process is repeated as the next heaviest organelle is removed
Give two advantages of an electron microscope
- High resolving power; The electron beam has a very short wavelength
- Beam can be focused; As electrons are negatively charged, the beam can be focused using electromagnets
Why is a ‘near’ vacuum needed for an electron microscope to work effectively?
- Electrons are absorbed or deflected by molecules in the air
What are the two types of electron microscope?
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
What is the TEM microscope?
- Consists of an electron gun
- Beam of electrons can be focused onto specimen with an elecctromagnet
What are the main limitations of the TEM?
- Living organisms cannot be processed; Vacuum is necessary and water based organisms would boil
- Staining process is complex; Image is in black and white
- Specimen must be extremely thin
- May contain artefacts; Depends on process and accuracy of specimen preparation eg dehydration of the specimen
