MICROSCROPY NOTES

 

Preparation of samples

  • Fixation: Chemicals preserve material in a life like condition. Does not distort the specimen.
  • Dehydration: Water removed from the specimen using ethanol. Particularly important for electron microscopy because water molecules deflect the electron beam which blurs the image.
  • Embedding: Supports the tissue in wax or resin so that it can be cut into thin sections.
    Sectioning Produces very thin slices for mounting. Sections are cut with a microtome or an ulramicrotome to make
    them either a few micrometres (light microscopy) or nanometres
    (electron microscopy) thick.
  • Staining: Most biological material is transparent and needs staining to increase the contrast between different structures. Different stains are used for different types of tissues. Methylene blue is often used for animal cells, while iodine in KI solution is used for plant tissues.
  • Mounting: Mounting on a slide protects the material so that it is suitable for viewing over a long period.

Magnification and Resolution

Magnification is how much bigger a sample appears to be under the microscope than it is in real life.

Overall magnification = Objective lens x Eyepiece lens

Resolution is the ability to distinguish between two points on an image.

  • The resolution of an image is limited by the wavelength of radiation used to view the sample.
  • This is because when objects in the specimen are much smaller than the wavelength of the radiation being used, they do not interrupt the waves, and so are not detected.
  • The wavelength of light (min. – violet is 400nm) is much larger than the wavelength of electrons, so the resolution of the light microscope is a lot lower.
  • The actual resolution is often half the size of the wavelength of radiation used. Thus, for the light microscope the maximum resolution is about 200nm.
  • In other words, if two objects in the specimen are closer than 200nm in real life, then they will only show up as one object on the image.
  • Using a microscope with a more powerful magnification will not increase this resolution any further. It will increase the size of the image, but objects closer than 200nm will still only be seen as one point.

 

Transmission and Scanning Electron Microscopes

  • Transmission electron microscopes pass a beam of electrons through the specimen. The electrons that pass through the specimen are detected on a fluorescent screen on which the image is displayed.
  • Thin sections of specimen are needed for transmission electron microscopy as the electrons have to pass through the specimen for the image to be produced.

 

  • Scanning electron microscopes pass a beam of electrons over the surface of the specimen in the form of a ‘scanning’ beam.
  • Electrons are reflected off the surface of the specimen as it has been previously coated in heavy metals.
  • It is these reflected electron beams that are focussed of the fluorescent screen in order to make up the image.
  • Larger, thicker structures can thus be seen under the scanning electron microscope as the electrons do not have to pass through the sample in order to form the image.
  • However the resolution of the scanning electron microscope is lower than that of the transmission electron microscope.

     

Comparison of the light and electron microscope

 

Light microscope

 

 

Electron microscope

Cheap to purchase (£100 – 500)

Expensive to buy (over £ 1 000 000).

Cheap to operate.

Expensive to produce electron beam.

Small and portable.

Large and requires special rooms.

Simple and easy sample preparation.

Lengthy and complex sample prep.

Material rarely distorted by preparation.

Preparation distorts material.

Vacuum is not required.

Vacuum is required.

Natural colour of sample maintained.

All images in black and white.

Magnifies objects only up to 2000 times

Magnifies over 500 000 times.

 

Basic Principles of Light and Electron Microscopy

Light Microscopy

  • Light is produced from either an internal or external light source and passes through the iris diaphragm, a hole of variable size which controls the amount of light reaching the specimen.
  • The light then passes through the condenser which focuses the light onto the specimen.
  • The slide is held on the stage at 90 degrees to the path of light which next travels through the specimen.
  • The objective lens magnifies the image of the specimen before the light travels through the barrel of the microscope.
  • The light finally passes through the eyepiece lens and into the viewer’s eye which sends impulses to the brain which in turn interprets the image.

Electron Microscopy

  • A negatively charged platinum metal electrode (the cathode) emits a beam of high velocity negatively charged electrons.
  • The electromagnets on the side of the barrel focus the beam of electrons on the specimen in the same way that the glass lenses on a light microscope focus the beams of light.
  • The specimen is introduced via an air lock so as to maintain the internal vacuum conditions.
  • The transmitted or reflected beam of electrons, depending on type of microscope are focused by the electromagnets onto a fluorescent screen to produce the image which is then viewed by the operator.

 

 

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