Microscopes Flashcards
What is the formula for magnification?
Size of Image / Size of Real Object
What is resolution?
Resolution/Resolving Power, is the minimum distance apart that two objects can be in order for them to appear as separate items.
What is cell fractionation?
Where cells are broken up, and the different organelles they contain are separated out.
Why is the solution kept cold?
To reduce enzyme activity that might break down the organelles.
Why is the solution kept isotonic?
To keep the same water potential as the original tissue, it is to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
Why is the solution buffered?
So that the pH does not fluctuate, any changes in pH could alter the structure of the organelles, or effect the functioning of the enzymes.
What are the two stages of cell fractionation?
- Homogenation
* Ultracentrifugation
What is the process of homogenation?
- Cells are broken up by a homogeniser
- The organelles are released from the cell, and the resultant fluid containing it is called the homogenate.
- It is filtered to remove debris and complete cells.
What is the process of ultracentrifugation?
- Tube of filtrate is placed in the centrifuge and spun at a slow speed
- Heaviest organelles, the nuclei are forced to the bottom of the tube, where they form a thin sediment.
- The fluid at the top of the tube, the supernatant, is removed, leaving just the sediment of nuclei.
- Supernatant transferred to another tube and spun in the centrifuge at a faster speed than before.
- Next heaviest organelles, are forced to the bottom of the tube,.
- Process repeated until desired organelle forms a pellet at the bottom.
Why were electron microscopes used over light microscopes?
- Electron beams have a very short wavelength, and the microscopes can therefore resolve objects well, high resolving power.
- Electrons are negatively charged, so the beam can be focused using electromagnets.
What are the two types of electron microscope?
- Transmission Electron Microscope
* Scanning Electron Microscope
How do Transmission electron microscopes work?
- Electromagnets are used to focus a beam of electrons which is transmitted through the specimen.
- Denser parts of the specimen absorb more electrons, which makes them darker on the image you end up with.
Why can the true resolving power not be achieved in practice?
- Difficulties preparing the specimen limit the resolution that can be achieved.
- A higher energy electron beam is required, this may destroy the specimen.
What are the limitations of TEMs?
- A vacuum is required, therefore living specimens cannot be observed.
- A complex staining process is required, and the image is not in colour.
- Specimen must be extremely thin.
- The image may contain artefacts
What are artefacts?
- Things that result from the way the the specimen is prepared, artefacts are not a part of the specimen and may appear on the finished image.
- It results in difficulties telling whether a specific item is an artefact or a part of the specimen.
