2A - Analysis of cell components Flashcards
What is magnification?
How many times bigger the image is compared to the object.
What is the equation for magnification?
Magnification = Image size/object size
What is resolution?
The minimum distance apart that 2 objects can be in order for them to appear as separate items.
What is cell fractionation?
The process where cells are broken up and the different organelles they contain are separated out.
What has to happen before cell fractionation can begin?
The tissue is placed in a cold, buffered, isotonic solution.
Why is the tissue placed in a cold solution before cell fractionation?
To reduce enzyme activity which might break down the organelles.
Why is the tissue placed in an isotonic solution before cell fractionation?
To prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
Why is the tissue placed in a buffered solution before cell fractionation?
So that pH doesn’t fluctuate. Any change in pH could alter the structure of the organelles or affect the functioning of enzymes.
What are the 2 stages of cell fractionation?
Homogenisation
Ultracentrifugation
What happens in homogenisation?
Cells are broken up by a homogeniser (blender) which releases the organelles from the cell. The resultant fluid, known as the homogenate, is then filtered to remove any complete cells and large pieces of debris.
What is ultracentrifugation?
The process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge. This spins tubes of homogenate at very high speed in order to create a centrifugal force.
What happens in ultracentrifugation of animal cells?
The tube of filtrate is placed in the centrifuge and spun at a low speed.
The heaviest organelles, nuclei, are forced to the bottom of the tube, where they form a thin sediment or pellet.
The fluid at the top of the tube (supernatant) is removed, leaving just the sediment of nuclei.
The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before.
The next heaviest organelles, the mitochondria, are forced to the bottom of the tube.
The process is continued in this way so that, at each increase in speed, the next heaviest organelle is sedimented and separated out.
What are the advantages of electron microscopes?
Electron beam has a very short wavelength and the microscope can therefore resolve objects well.
Electrons are negatively charged so the beam can be focused using electromagnets.
Why do you have to use electron microscopes in a vacuum?
Because electrons are absorbed or deflected by the molecules in the air .
What are the 2 types of electron microscopes?
transmission electron microscope (TEM)
scanning electron microscope (SEM)
What is magnification controlled by?
The power of the lens used.
What is resolution controlled by?
The wavelength of the illumination used.
What are the benefits of using transmission electron microscopes?
high magnification
high resolution
focuses using a condenser electromagnet
produces 2D photomicrograph
What are the limitations of using transmission electron microscopes?
can't look at living cells must be in a vacuum must cut a section/thin specimen complicated prep may create artefact doesn't produce a colour image
What are the benefits of using scanning electron microscopes?
high magnification medium resolution specimen doesn't need to be thin focuses using a condenser electromagnet produces 3D image using computer analysis
What are the limitations of using scanning electron microscopes?
same as TEM can't look at living cells must be in a vacuum complicated prep may create artefact doesn't produce a colour image
What is the magnification and resolution like in a light microscope?
low
Can you use a live specimen under a light microscope?
yes
Is the image produced by a light microscope in colour?
yes
What is the eyepiece graticule and what does it do?
A glass disc that is placed in the eyepiece which is used to measure the size of objects.
How do you calibrate the eyepiece graticule?
Using a stage micrometer.
Who developed electron microscopy?
Robert Hooke.
