Microscopes

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  • Created by: yott33
  • Created on: 09-02-16 19:08

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.

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Calculating Magnification

Formula: Image size ÷ object size. 

You might have to rearrange the formula to work out the image size or object size.

Example:

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

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Light Microscopes

  • 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
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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
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Electron Microscopes

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.

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Compare Magnification and Resolution

Maximum Resolution

Light = 0.2 micrometers (low)

TEM = 0.0002 micrometers (high)

SEM = 0.002 (medium)

Maximum Magnification

Light = X1500

TEM = X1 000 000 + 

SEM = Less than X500,000

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Staining Samples

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.

Light Microscopes

  • 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

Electron Microscopes

  • Objects are dipped in a solution of heavy metal e.g. lead.
  • The metal ions scatter the elctrons, again creating contrast.
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Preparing a Microscope Slide

Dry Mount

  • 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

Wet Mount

  • 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
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