Methods of studying cells Flashcards
Magnification
The degree to which the size of an image is larger than the object itself
Resolution
The ability to distinguish between 2 points.
- the minimum distance apart two objects can be to be seen as separate items
Conditions for electron microscopes
-Specimen must be dead and viewed in a vacuum
- staining using metal dyes is always necessary
-Images are black and white
Transmission electron microscope
Consists of electron gun that produces a beam that is focused onto the specimen by a condenser electromagnet
Scanning electron microscope
Directs a beam of electrons on to the surface of the specimen in regular patterns and the electrons are scattered by the specimen building up a 3-D image
Give one advantage of using a TEM compared with a SEM
-Higher resolution
-view internal structures
What is the equation for magnification
Magnification= size of image/ size of real object
Why do light microscopes have poor resolution
long wavelengths of light
What are the advantages to using an electron microscope
-the electron beam has a very short wavelength and therefore can resolve objects well
- 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
What type of images do TEM microscopes produce
2-D
What type of images do SEM microscopes produce
3-D
What are the limitations of a TEM microscope
-specimen must be extremely thin
-complex staining process is required
-whole system must be in a vacuum
What is the resolving power of a light microscope
0.2Um
What is the resolving power of an electron microscope
0.1nm
How do you measure the size of an object using a light microscope
eyepiece graticule
How do you calibrate an eyepiece graticule
using a stage micrometer.
when the eyepiece graticule and stage micrometer are lined up it’s possible to calculate the length of divisions on the eyepiece graticule
Homogenisation
cells are broken up by a homogeniser to release the organelles from the cell. The homogenate is filtered out to remove large debris
What is cell fractionation
The process where cells are broken up and the different organelles they contain are separated out
What are the two stages of cell fractionation
- Homogenation
-ultracentrifugation
What is Ultracentrifugation
The process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge
Why is the solution cold
to reduce enzyme activity
Why is the solution buffered
to reduce pH change that might damage organelles or affect the functioning of enzymes
Why is the solution isotonic
To prevent osmotic changes that may cause the cells to shrink or burst
What is the process of Ultracentrifugation
spin filtrate from homogenisation at slow speed in centrifuge, heavy organelles (nuclei) fall to the bottom - fluid from the top (supernatant) removed leaving nuclei as a pellet - spin supernatant at higher speed, next heaviest organelles (mitochondria) fall to the bottom - repeat, spin supernatant at higher speed, next heaviest organelle falls to the bottom etc
Describe how a sample of chloroplasts could be isolated from leaves.
- Break open cells/tissue and filter
- In cold, same water potential/concentration, pH controlled solution;
- Centrifuge/spin and remove nuclei/cell debris;
- (Centrifuge/spin) at high(er) speed, chloroplasts settle out;
