Methods of Studying Cells and The Electron Microscope

Bee Brittain
Flashcards by Bee Brittain, updated more than 1 year ago
Bee Brittain
Created by Bee Brittain about 5 years ago
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AS - Level Biology (3 - Cell Structure) Flashcards on Methods of Studying Cells and The Electron Microscope, created by Bee Brittain on 04/07/2016.

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Question Answer
Define the term, Resolution Resolution is how detailed the image is and how well a microscope can distinguish two points that are close together
Define the term, Magnification Magnification is how many bigger the image is than the actual specimen
What is the formula to work out magnification? Magnification = Image size / Actual Size
Optical microscopes form an image using what? Beam of light
The maximum useful magnification of a light microscope is x_____ x1500
The maximum resolution of a light microscope is what? Give some organelles you CAN'T see using an optical microscope. About 0.2micrometres You can't some organelles including ribosomes, ER and lysosomes. Mitochondria may be seen, however they won't be in perfect detail.
To view a specimen under an optical microscope, what much you prepare? A temporary mount
What is the first step in preparing a temporary mount? Add a small drop of water on to the slide and using tweezers place a thin section of your specimen on top of the water droplet
After the specimen is on the slide, what happens next when preparing a temporary mount? A drop of stain is added to highlight objects in the cell. E.g/ Iodene is used to highlight starch
What is the final step of preparing a temporary mount? Carefully place a slide over over the specimen, try to 'roll' it on to decrease the risk of getting air bubbles - these may obstruct your view when trying to view the specimen
What does TEM stand for? Transmission Electron Microscope
A TEM uses what in order to produce an image? Electron beam that if focused by electromagnets
What type of image does a TEM produce? A 2D, black and white image
The electron beam has a very short wavelength, what does this mean for a TEM? It has a very high resolving power of about 0.1nm
The specimen used in a TEM has to be very thin, why? In a transmission electron microscope the electrons in the electron beam has to pass through the specimen, which is why it has to be thin otherwise the image produced wouldn't be clear as the electrons would not have been able to pass through the whole specimen
TEM have a high resolution, what does this mean in terms of organelles? They can see organelles smaller than those you see under an optical microscope. Due to a TEM's high resolution, you can also see internal structure of organelles, e.g/ chloroplasts
The image produced on a TEM is black and white or, grey-scale. Why is the image produced a grey-scale image? Its grey-scale because more dense parts of the specimen, absorb more electrons than the less dense parts when the electrons pass through the specimen.
What does SEM stand for? Scanning Electron Microscope
Is the resolution of a SEM higher, or lower, than that of a TEM. Can you give the resolutions of both? The resolution of a SEM is lower than that of a TEM. SEM resolution: 20nm TEM resolution: 0.1nm
Describe a feature about the image a SEM produces It is 3D as the electrons hit the surface of the specimen, then reflect back.
Describe vaguely how an image from a SEM is produced The electron beam is scanned across the specimen. Electrons which are knocked off and reflected are gathered in a cathode ray tube to form the image
Electrons from a TEM pass through the specimen, where as electrons from a SEM only scan it. What does this mean about the thickness of a specimen from each microscope? It just means that the specimen from the TEM would have to be thinner than the specimen from the SEM
The system of both TEM's and SEM's are in a vacumn, what limitation does this make? The limitation is that any specimens view under an electron microscope much be dead
In order to show up different parts of the specimen, is has to undergo a complex staining process, what limitation does this make? It means that the specimen could possible be damaged or not in true colour
Preparation of specimens for electron microscopes is a complex process with many limitation and difficulties, what does this mean? It means that in the final image (the pictomicrograph) there may be foreign objects, providing a false image
There are 3 main steps of cell fractionation and ultracentrifugation, name them and give a sentance to what occurs in them. 1) Homogenisation - breaking up the cell 2) Filtration - filtering the cell and getting rid of the 'big bits' 3) Ultracentrifugation - Seperating the Organelles
Describe and explain the HOMOGENISATION step in cell fractionation. During homogenisation, the cells are broken up by a homogeniser (blender). This breaks up the plasma membrane and releases the cell's organelles into solution - called homogenate.
The homogenate solution should ISOTONIC, what does this mean and why is this a required condition for the homogenate? The solution should be isotonic. This means it should have the same concentration of chemicals as the cells being broken down. This ensures that the water potential is the same so the cell doesn't shrink or cell lysis doesn't occur
Another condition the homogenate should be is ICE-COLD. Explain why the solution needs to be so cold. The solution has to be ice cold to reduce the activity of the enzymes that break down organelles
The homogenate also has to be the correct pH, what can be added to ensure a steady pH and explain why it is needed. To enure pH is maintained and is steady, a buffer solution is added. This has to be added as otherwise, a change in pH could alter the shape of organelles.
The second step of cell fractionation and ultracentrifugation is filtration and getting rid of the 'big bits'. Describe how this works. The homogenate is filtered through a gauze to separate any large cell debris or tissue debris from the organelles. The organelles are much smaller than the debris and so they pass through the gauze
The final step of this process, is ultracentrifugation. Fill in the blanks: The tube of _______ is placed in the ________ and spun at a _____ speed. The heaviest __________ fall to the _______ of the test tube and form a thick _________. The tube of FILTRATE is placed in the CENTRIFUGE and spun at a LOW speed. The heaciest ORGANELLES fall to the BOTTOM of the test tube and form a thick SEDIMENT.
After being spun at a low speed, there is a fluid at the top of the test tube. What is this called? Supernatant
After the supernatant is removed, what happens to it? It is transferred to another test tube and spun in the centrifuge at a higher speed than before. Again, the next heaviest organelles (mitochondria) are forced to the bottom of the tube. This process is continued in this way so each organelle is separated.
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