Microscopy

Types

Light Microscope – uses a number of lenses to produce an image that can be viewed directly at the eyepiece. Light passes from a bulb under the stage, through a condenser lens and then through the specimen. This beam of light is passed through an objective lens (x4, x10, x40) and then the eyepiece lens (x10)

Transmission Electron Microscope (TEM) – uses electromagnets to focus a beam of electrons, which is transmitted through the specimen. Denser parts of the specimen absorb more electrons, which makes them look darker on the image you end up with creating a contrast.

Scanning Electron Microscope (SEM) – an electron beam is scanned across the specimen. The electrons don’t pass through the specimen, they bounce off and are detected at multiple detectors.

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Comparison

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Advantages and Disadvantages of Light

Advantages

  • Wide range of specimens can be observed
  • Specimens can be alive
  • Specimens can be whole or embedded in wax then sectioned

Disadvantages

  • Non-coloured specimens must be stained for specific organelles or molecules
  • It has a low resolution so does not give detailed information of the object looked at
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Advantages and Disadvantages of Electron

Advantages

  • Produces detailed images of the structures inside cells
  • SEM produces detailed 3D images showing the contour of cells

Disadvantages

  • Electron beams deflected in air, so sample must be in a vacuum
  • Samples must be dead
  • Extremely expensive
  • A large piece of equipment
  • Use requires a high degree of skill and training
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Stains

Sometimes the object is view as transparent making it hard to see anything because the light rays and electrons just pass through the object but this can be avoided by staining

Different stains are used to make different things visible 

For example in light microscopes:

  • Eosin is used to stain cell membranes
  • Methylene blue stains DNA

But electron microscopes are different:

  • Electron microscopes are stained with heavy metals (like lead) to scatter the electrons and create contrast
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Eyepiece Graticule and Stage Micrometer

Sometimes to know the size of specimen eyepiece graticules and stage micrometers are used similarly to rulers

The graticule is fitted onto the eyepiece and is like a transparent ruler with no units and a stage micrometer is placed onto the stage, this has an accurate scale in order to work out the value of the divisions on the eyepiece graticule at a particular magnification

Once the micrometer is removed and is replaced with the specimen the size can be measured

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Definitions, Conversions and Formulas

  • Magnification = Number of times larger an image appears compared to the real size of the object
  • Resolution= The ability to distinguish two separate points as distinct from each other

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