Methods of Studying Cells
Biology: AQA A Level, topic 2.1.6
Magnification and Resolution
Two parameters that are important in microscopy are magnification and resolution.
Magnification
- Magnification is the process of enlarging an object in appearance.
- The image size is how big the object appears to be in a picture or drawing, which will be in milimeters (mm).
- The actual size is often given in micrometers (µm) - units must be converted so that they are the same.
Calculating magnification
- The equation for magnification is:
- Magnification = size of image ÷ size of real object
- E.g. A mitochondrion is 20 µm long. An image of the mitochondrion is measured as 20 mm long. What is the magnification?
- Magnification = 20,000 µm ÷ 20 µm = 1,000x
Resolution
- Resolution is the ability of a microscope to distinguish two adjacent structures as separate.
- The higher the resolution, the better the clarity and detail of the image.
Cell Fractionation
Cell fractionation separates organelles according to size to allow them to be studied in an electron microscope. The steps involved are:
1) Homogenisation
- The tissue sample is homogenised using a blender to break the cells.
- The tissue sample must be kept in specific conditions:
- Ice cold (reduces enzyme activity that might damage organelles).
- Isotonic solution (prevents osmosis that could shrink or burst organelles).
- No osmosis takes place in isotonic solution.
- Buffered solution (keeps pH constant and avoids damaging the protein structures).
2) Filtration
- The tissue sample is filtered into tubes through a gauze.
- The gauze separates larger components from the small, organelles.
- The organelles are filtered into tubes to be fractionated using ultracentrifugation.
3) Ultracentrifugation
- The samples are spun at a low speed in a centrifuge.
- Each tube must be balanced with another tube directly opposite for the centrifuge to work properly.
- Centrifugation separates the sample into fractions.
- Heavier organelles are forced to the bottom of the tube.
- Lighter organelles move towards the top.
- Cell debris (e.g. cell walls) forms a pellet at the bottom of the tube, leaving the supernatant (a liquid) above it that contains the organelles.
4) Ultracentrifugation
- The supernatant is poured off and centrifuged at a higher speed to separate the next heaviest organelles (the nuclei).
- This is repeated at increasingly higher speeds to separate each fraction.
5) Order of fractionation
- The process of repeated ultracentrifugation produces fractions of cell organelles from heaviest to lightest. This order is:
- Nucleus.
- Chloroplasts.
- Mitochondria.
- Lysosomes.
- Endoplasmic reticulum.
- Ribosomes.
Test yourself on Methods of Studying Cells
Which of the following statements regarding resolution and magnification are true?
Stages in Cell Fractionation
Which of these organelles is heaviest?
Which of the following statements about cell fractionation is true?