- Created by: rosiebaxter.x
- Created on: 30-08-18 17:08
- How many times bigger the image is when compared to the object.
Magnification = Size of Image / Size of Actual Object
- 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.
- 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.
- An electron gun produces a beam of electrons that is focused onto the specimen by a condenser electromagnet.
-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)
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
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.
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
- 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.
-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.