A3. Analysis of Cell Components Flashcards
What is magnification and how is it calculated?
Magnification is how much bigger the image is than the specimen (the sample you’re looking at). Magnification = image size/actual size
What is resolution?
Resolution is how detailed the image is. More specifically, it’s how well a microscope distinguishes between two points that are close together. If a microscope lens can’t separate two objects, then increasing the magnification won’t help.
Types of microscope - Optical (light) microscopes
What do they use?
What can they not see?
What can they see?
They use light to form an image. They have a maximum resolution of about 0.2 micrometres (um). This means you can’t use an optical microscope to view organelles smaller than 0.2 μm. That includes ribosomes, the endoplasmic reticulum and lysosomes. You may be able to make out mitochondria–but not in perfect detail. You can also see the nucleus. The maximum useful magnification of an optical microscope is about x 1500.
Electron microscopes
What do they use?
What do they make?
They use electrons to form an image. They have a higher resolution than optical microscopes, so give a more detailed image (and can be used to look at more organelles). They have a maximum resolution of about 0.0002 micrometres (um). (About 1000 times higher than optical microscopes.) The maximum useful magnification of an electron microscope is about x 1 500 000. Electron microscopes produce black and white images, but these are often coloured by a computer.
Transmission electron microscopes (TEMS)
What do they use?
What can they see?
TEMS use electromagnets to focus a beam of electrons, which is then transmitted through the specimen. Denser parts of the specimen absorb more electrons, which makes them look darker on the image you end up with. TEMs are good because they give high resolution images, so you see the internal structure of organelles like chloroplasts. But you’ve got to view the specimen in a vacuum, so they’re no good for looking at living organisms. They can also only be used on thin specimens.
Scanning electron microscopes (SEMs)
What do they use?
What can they see?
SEMS scan a beam of electrons across the specimen. This knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image. The images you end up with show the surface of the specimen and they can be 3-D. SEMS are good because they can be used on thick specimens, but they give lower resolution images than TEMs.
Figure 4: Comparison table of TEM and SEM features.
Preparing microscope slides in 4 steps
- Start by pipetting a small drop of water onto the centre of the slide.
- Then use tweezers to place a thin section of your specimen on top of the water drop. (Your specimen needs to let light through it for you to be able to see it clearly under the microscope-so if you’ve got quite a thick specimen, you’ll need to take a thin slice to use on your slide).
- Add a drop of a stain. Stains are used to highlight objects in a cell.
- Finally, add the cover slip (a square of clear glass or plastic that protects the specimen). To do so, stand the slip upright on the slide, next to the water droplet. Then carefully tilt and lower it so it covers the specimen. Try not to get any air bubbles under there (see below) - they’ll obstruct your view of the specimen.
Microscope artefacts
What are artefacts and what can they be?
Where are they common?
How to see if something is an artefact?
Artefacts are things that you can see down the microscope that aren’t part of the cell or specimen that you’re looking at. They can be anything from bits of dust, air bubbles and fingerprints, to inaccuracies caused by squashing and staining your sample. Artefacts are usually made during the preparation of your specimen and shouldn’t really be there at all.
Artefacts are especially common in electron micrographs because specimens need a lot of preparation before you can view them under an electron microscope.
The first scientists to use electron microscopes could only distinguish between artefacts and organelles by repeatedly preparing specimens in different ways. If an object could be seen with one preparation technique, but not another, it was more likely to be an artefact than an organelle.
Cell fractionation - There are three steps to this technique:
- Homogenisation-breaking up the cells
- Filtration - getting rid of the big bits
- Ultracentrifugation-separating the organelles
- Homogenisation-breaking up the cells
How can it be done?
What does it do?
What must the solution be?
Why must the solution be this way?
Homogenisation can be done in several different ways, e.g. by vibrating the cells or by grinding the cells up in a blender. This breaks up the plasma membrane and releases the organelles into solution.
The solution must be kept ice-cold, to reduce the activity of enzymes that break down organelles. The solution should also be isotonic-this means it should have the same concentration of chemicals as the cells being broken down, to prevent damage to the organelles through osmosis. A buffer solution should be added to maintain the pH.
- Filtration - getting rid of the big bits
Next, the homogenised cell solution is filtered through a gauze to separate any large cell debris or tissue debris, like connective tissue, from the organelles. The organelles are much smaller than the debris, so they pass through
the gauze.
- Ultracentrifugation-separating the organelles
After filtration, you’re left with a solution containing a mixture of organelles. To separate a particular organelle from all the others you use ultracentrifugation: (3 steps)
The organelles are separated in order of mass (from heaviest to lightest) -this order is usually:…
- The cell fragments are poured into a tube. The tube is put into a centrifuge (a machine that separates material by spinning) and is spun at a low speed. The heaviest organelles, like nuclei, get flung to the bottom of the tube by the centrifuge. They form a thick sediment at the bottom- the pellet. The rest of the organelles stay suspended in the fluid above the sediment-the supernatant.
- The supernatant is drained off, poured into another tube, and spun in the centrifuge at a higher speed. Again, the heaviest organelles form a pellet at the bottom of the tube. The supernatant containing the rest of the organelles is drained off and spun in the centrifuge at an even higher speed.
- This process is repeated at higher and higher speeds, until all the organelles are separated out. Each time, the pellet at the bottom of the tube is made up of lighter and lighter organelles.
The organelles are separated in order of mass (from heaviest to lightest) -this order is usually: nuclei, then mitochondria, then lysosomes, then endoplasmic reticulum, and finally ribosomes. In plant cells, the chloroplasts come out after the nuclei, but before the mitochondria.
