Methods of Studying Cells

Magnification

- How many times bigger the image is when compared to the object.

Magnification = Size of Image / Size of Actual Object

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Resolution

- The minimum distance apart that two objects can be, in order for them to appear as separate items. 

- Resolving power depends on wavelength or form of radiation used.

- The larger the wavelength, the worse the resolution.

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Light Microscope

- Uses a beam of light which travels through a thin specimen.

- 2 glass lenses focus the light.

- Staining needs to be used.

- Resolving power = 200nm

- Magnification = x1500

Advantages - can use a live specimen.

                   - relatively quick preparation / little cost.

                   - in colour.

Disadvantages - poor resolution due to large wavelength of light.

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Electron Microscope

- An electron gun produces a beam of electrons that is focused onto the specimen by a condenser electromagnet.

Advantages:

-The electron beam has a very short wavelength so it can resolve objects of up to 0.1nm (2000x better than a light microscope)

-As electrons are negatively charged, the beam can be focused using electromagnets.

There are two different types: 

-Transmission Electron Microscope (TEM)

-Scanning Electron Micrscope (SEM)

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Transmission Electron Microscope

- The electron beam is passed through a thin section of the specimen and creates a photomicrograph as the electrons reach the other side.

- The thicker parts of the specimen absorb the electrons and appear dark whereas the thinner parts allow the electrons to go through and appear lighter.

-Resolving power = 0.1 nm

-Magnification = x2 000 000

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Limitations of the TEM

-The whole system must be in a vacuum, so living specimens cannot be observed.

-A complex staining process is required. (costs money and time)

-The specimen must be very thin.

-As the specimen has to be thin it only allows us to get a 2D image. (However we can now take a series of these 2D sections and build a 3D one but is a slow and complicated process)

-The image may contain artefacts (unnatural parts of the specimen that may occur from the way the specimen was prepared).

-Resoloving power of 0.1nm cannot always be acheived due to difficulties preparing the specimen or a higher energy beam is needed which may destroy the specimen.

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Scanning Electron Microscope

-Directs the beam onto the surface of the specimen which is passed back and forth in a regular pattern. 

-The pattern of scattered electrons depends on the contours of the specimen which builds a 3D image.

- Specimen does need to be so thin as the electrons dont penetrate the cell.

- Resolving power = 20nm.

- Magnification = x1 500 000

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Measuring Cells

- Eyepiece Graticule: A disc engraved with a scale (in the lense).

--> It needs to be calibrated first with a stage micrometre (ruler).

--> You have to see how many graticules go into 1mm (1000um) then work out how many micrometres is in one graticule.

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Cell Fractionation

-Tissue is cut into pieces and put in a 

COLD (to reduce enzyme activity which breaks down organelles),

BUFFERED (to maintain a constant pH),

ISOTONIC (to prevent organelles bursting/shrinking from osmotic gain/loss) SOLUTION.

-The tissue is HOMOGENISED (blended).

-The homogenate is FILTERED to REMOVE DEBRIS.

-Filtrate is CENTRIFUGED at a low speed.

-SUPERNATENT is analysed for content, then is decanted and re-centrifused at higher speeds until desired organelle is separeted.

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