Magnification and Resolution
Magnification = how much BIGGER the image is than the specimen itself. It's calculed using the formula:
Magnification = image size ÷ object size
Resolution = how DETAILED the image is. It is how well a microscope can distinguish between two points that are close together.
Formula: Image size ÷ object size.
You might have to rearrange the formula to work out the image size or object size.
A magnified image is 5mm wide. Your specimen is 50 micrometers wide.
1. Get everything into the same units: 1mm = 1000 micrometers
2. So 50 micorometers divided by 1000 = 0.05mm
3. 5 ÷ 0.05 = X100
- Use light
- Lower resolution than electron microscopes
- Maximum resolution of approx. 2 micrometers.
- Usually used to look at whole cells or tissues.
- Maximum useful magnification = approx. X1500
Laser Scanning Confocal Microscopes
- Uses laser beams to scan a specimen, which is usally tagged with a fluorescent dye.
- Laser causes dye to give off light (fluoresce).
- The light is then focused through a pinhole onto a detector.
- Detector hooked up to a computer which generates an image. Image can be 3D.
- The pinhole means that any out-of-focus light is blocked = clearer image
- Can be used to look at objects at different depths in thick specimens
They use electrons to form an image. They have a higher resolution to give more detailed images. Two kinds.
Transmission Electron Microscope (TEM)
Electromagnets to focus a beam of electrons, which is transmitted through the specimen.
Denser parts absorb more electrons = look darker on image
Can provide high res. images but can only be used on thin specimen
Scanning Electron Microscope (SEM)
Scan a beam of electrons across the specimen.
Knocks off electrons from specimen which are gathered in a cathode ray tube to form an image.
Shows surface of specimen and can be 3D. Lower resolutions than TEM.
Compare Magnification and Resolution
Light = 0.2 micrometers (low)
TEM = 0.0002 micrometers (high)
SEM = 0.002 (medium)
Light = X1500
TEM = X1 000 000 +
SEM = Less than X500,000
In light microscopes and TEMs, the beam of light/electrons passes through the object being viewed - an image is produced because som parts of the object absorb more light/electrons than others. If the object being viewed is transparent, it makes the whole thing look white so you have to stain it.
- Using a dye e.g. methylene blue and eosin
- The stain is taken up by some parts of the object more than others - the contrast makes the different parts show up
- Different stains are used to make different things show up - eosin = cell cytoplasm
- More than one stain can be used at once
- Objects are dipped in a solution of heavy metal e.g. lead.
- The metal ions scatter the elctrons, again creating contrast.
Preparing a Microscope Slide
- If you have a thick specimen, you'll need to use a thin slice to use on your slide
- Use tweezers to pick up specimen and put it in the middle of a clean slide
- Put a cover slip on top
- Pipette a small drop of water onto the slide
- Use tweezers to place the specimen on the water drop
- Stand the cover slip upright on the slide next to the water droplet then carefully tilt and lower it so it covers the speciment. Try to not get air bubbles.
- Once the cover slip is in position, you can add a stain. Put a drop of stain next to one edge of the cover slip then put a bit of paper towel next to the opposite edge - the stain will get drawn under the slip across the specimen