Studying Cells Flashcards
(39 cards)
What is the equation for calculating magnification
Magnification= image size/actual size
Define resolution/resolving power
The resolution of a microscope is the minimum distance apart that two objects can be in order for them to appear as separate items.
What effect does a high resolution have on an image
The greater the resolution, the greater the clarity and precision of the image.
What is a microscopes limit of resolution?
The limit of resolution is the point where before reaching it, increasing the microscopes magnification will reveal more detail in an image but after it, increasing magnification will increase the size of the image but will not reveal more detail- the image will just become more blurred.
What is cell fractionation
The process where cells are broken up and the different organelles that they contain are separated out
What is magnification
The scale/number of times an image has been enlarged from its actual size to the size of the image.
What are the two stages in cell fractionation
Homogenation
Ultracentrifugation
What must happen to the tissue before cell fractionation can begin
It must be placed in a cold, isotonic buffer solution.
Why must the solution that the tissue is placed in before cell fractionation be cold
To reduce enzyme activity that might break down the organelles.
Why must the solution that the tissue is placed in before cell fractionation be buffered
It must be buffered to ensure that the PH does not fluctuate as a change in PH could alter the structure of organelles and affect how enzymes function.
Why must the solution that the tissue is placed in before cell fractionation be isotonic
The solution must have the same water potential as the tissue to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
Describe the first stage of cell fractionation- homogenation
The cells are broken up by a homogeniser (blender). The resultant fluid (homogenate) is filtered to remove any complete cells and large pieces of debris.
Briefly describe the second stage of cell fractionation- ultracentrifugation
Ultracentrifugation is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge which spins the solution at different speeds to separate the different organelles.
Describe the process of ultracentrifugation
- The tube of filtrate is placed in the centrifuge and spun at a slow speed
- The heaviest organelles- the nuclei are forced to the bottom of the tube where they form a thin layer of sediment or a 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 to form a pellet or thin layer of sediment.
- This process is continued so that at each increase in speed, the next heaviest organelle is sedimented and separated out.
Briefly describe each step in the whole process of cell fractionation.
1) Tissue is cut into pieces in cold conditions in a buffered, isotonic solution
2) The tissue is homogenised to release the organelles from the cell
3) The homogenate formed is filtered
4) the filtrate is centrifuges at a low speed
5) The supernatant is decanted and re-centrifuged at higher speeds until the desired organelle is separated out.
6) the contents of the supernatant can be analysed
At roughly what speed of centrifugation (revolutions per minute) is a nuclei pellet formed
1000
At roughly what speed of centrifugation (revolutions per minute) are mitochondria and chloroplast pellets formed
3,500
At roughly what speed of centrifugation (revolutions per minute) are lysosome and endoplasmic reticulum pellets formed
16,500
At roughly what speed of centrifugation (revolutions per minute) are ribosome pellets formed
100,000
What are the two main advantages of an electron microscope
1) An electron beam has a very short wavelength and so electron microscopes can resolve objects very well-they have a high resolving power.
2) As electrons are negatively charged they can be focused using electromagnets.
Why must a near-vacuum be created within the chamber of an electron microscope
To prevent the electrons from being absorbed or deflected by molecules in the air
What are the two types of electron microscope
Scanning electron microscope
Transmission electron microscope
What are the main disadvantages/limitations of a transmission electron microscope
- The whole system must be in a vacuum and therefore living specimens cannot be observed.
- A complex ‘staining’ process is required and even then the image is not in colour.
- the specimen must be extremely thin to allow the electrons to penetrate and so the result is a 2D image.
- The image may contain artefacts which are abnormalities as a result of specimen preparation or due to the irradiation from the electron beam.
Why do the artefacts seen on an image created by an electron microscope (TEM & SEM) cause problems
Artefacts may appear on the finished photomicrograph but are not part of the natural specimen therefore it is difficult to tell if what we see on the photomicrograph really exists in that form.
