2.1.6

# Methods of Studying Cells

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## 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.