4991894
Description
Mind Map by Bee Brittain, updated more than 1 year ago
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Created by Bee Brittain
about 8 years ago
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3.1 & 3.2 Methods of studying
Cells and The Electron Microscope
- Optical Microscopes
- Use light to form an image
- Maximum resolution of about 0.2micrometres
- So you can't use an optical microscope to view
organelles smaller than 0.2 micrometres. This
includes ribosomes, ER and lysosomes. You may
see mitochondria, but not in peprfect detail.
- So you can't use an optical microscope to view
organelles smaller than 0.2 micrometres. This
includes ribosomes, ER and lysosomes. You may
see mitochondria, but not in peprfect detail.
- Maximum useful magnification of about x1500
- Using a slide to view specimens
- 1) add a small drop of water onto
the slide then use tweezers to place
a thin section of your specimen on
top of the water drop
- 2) Add a drop of stain to highlight
objects in the cell (e.g Iodene is used
for starch)
- 3) Finally, add a cover slip. Careful for air bubbles!
- 1) add a small drop of water onto
the slide then use tweezers to place
a thin section of your specimen on
top of the water drop
- Use light to form an image
- Transmission Electron Microscope (TEM)
- Use electromagnets to focus a beam on
electrons which is then transmitted
through the specimen
- Electron beam has a very short wavelength
- High resolving power of 0.1nm
- High resolving power of 0.1nm
- Electron beam has a very short wavelength
- Denser parts of specimen absorb more electrons = darker on image
- High resolution images = see internal
structure of organelles such as
chloroplasts
- Only can be used on thin specimens
- Limitations
- Whole system is in a vacumn = specimens must be dead
- Specimen has to undergo a complex staining process = can be damaged, not true colour.
- Specimen has to be extremely thin
- Image may contain artefacts ( things resulting from the way the
specimen is prepared) = foreign objects on end photomicrograph
- Whole system is in a vacumn = specimens must be dead
- Use electromagnets to focus a beam on
electrons which is then transmitted
through the specimen
- Scanning Electron Microscope (SEM)
- Scan a beam of electrons across the specimen
- knocks off electrons from the specimen which
are gathered in a cathode ray tube to form an
image
- knocks off electrons from the specimen which
are gathered in a cathode ray tube to form an
image
- Images show surface of specimen and so they can be 3D
- They have lower resolution than a TEM
- 20nm
- 20nm
- The Limitations of a SEM are very similar to a
TEM, however the specimen doesn't need to be
extremely thin as electrons do not penetrate it
- Scan a beam of electrons across the specimen
- Magnification is Size
- Magnification = Image size/Actual size
- Magnification is how much bigger the image is than the specimen
- Magnification = Image size/Actual size
- Resolution is detail
- Resolution is how detailed the image is, and how well a microscope
distinguishes between two points that are close together
- Resolution is how detailed the image is, and how well a microscope
distinguishes between two points that are close together
- Cell Fractionation and Ultracentrifugation
- If you want to look at some organelles under an electron microscope,
you'd first need to separate them from the rest of the cell = cell
fractionation.
- 1) Homogenisation - Breaking up the cells
- Cells are broken up by a homogeniser (blender). This breaks up the plasma
membrane and releases the organelles into solution. The solution is called
homogenate.
- Solution has to be ice-cold =
reduce activity of enzymes that
break down organelles
- Solution has to be Isotonic = same concentration of
chemicals as the cells being broken down/same water
potential as tissue to prevent organelles bursting/shrinking.
- A buffer solution should also
be added to homogenate to
maintain pH as a change in pH
could alter the structure of the
organelles
- Solution has to be ice-cold =
reduce activity of enzymes that
break down organelles
- Cells are broken up by a homogeniser (blender). This breaks up the plasma
membrane and releases the organelles into solution. The solution is called
homogenate.
- 2) Filtration - Getting rid of the big bits
- The homogenate is filtered through a gauze to seperate any large
cell debris or tissue debris from the organelles
- The organelles are much smaller than the debris so they pass through the gauze
- The organelles are much smaller than the debris so they pass through the gauze
- The homogenate is filtered through a gauze to seperate any large
cell debris or tissue debris from the organelles
- 3) Ultracentrifugation - Separating the organelles
- Separated in a machine called the centrifuge
- The tube of filtrate is placed in the centrifuge and spun at low speed
- The heaviest organelles, the nuclei, are forced to the bottom of the tube and form thing sediment
- 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
- Next heaviest organelles (e.g mitochondria) forced to the bottom of the tube
- The process is continued in this way so with each increase in speed the next heaviest organelle is sedimented and separated
- The process is continued in this way so with each increase in speed the next heaviest organelle is sedimented and separated
- Next heaviest organelles (e.g mitochondria) forced to the bottom of the tube
- The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before
- Fluid at the top of the tube (supernatant) is removed, leaving just the sediment of nuclei
- The heaviest organelles, the nuclei, are forced to the bottom of the tube and form thing sediment
- Separated in a machine called the centrifuge
- 1) Homogenisation - Breaking up the cells
- If you want to look at some organelles under an electron microscope,
you'd first need to separate them from the rest of the cell = cell
fractionation.
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