B2.1.1

• How do light microscope work?

  Light microscopes work when a beam of light is passed through a specimen before being focused through an objective lens where it is magnified and then magnified again by the eyepiece lense before reaching the eye.
• What are the advantages of a light microscope?

  • Can observe living specimens
  • Easy to set up and use
  • cheap
  • small and portable
  • show true colour
• What are the disadvantages of a light microscope?

  • Low magnification (x1500)
  • Low resolution (0.2um)
• what is the magnification of a light microscope?

  x1500
• What is the resolution of a light microscope?

  0.2 um
• why does a light microscope have a low resolution?

  due to the diffraction of light as it passes through a specimen, structures closer than half the wavelength of light cannot be seen separately (0.2um)
• what is a tem?

  Transmission electron microscope
• how does a TEM work?

  • electromagnets 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
• what are the advantages of a TEM?

  • High magnification (x500,000)
  • High resolution (0.0002um)
  • detailed image of cell ultrastructure and organelles
• what are the disadvantages of a TEM?

  • Expensive
  • training required
  • only dead specimens
  • black and white (false colour)
  • specimens can be damaged by the electron beam
  • Complex prep process produces artefacts
• What is an artefact?

  a visible structural detail caused by processing a specimen which is not a feature of the specimen itself
• what is an SEM?

  scanning electron microscope
• how does an SEM work?

  a beam of electrons is sent cross the surface of a specimen and reflected electrons are collected
• What are the advantages of an SEM?

  • produces 3D images
  • high magnification (x200,000) but not as good as a TEM
  • high resolution (0.002um) but not as good as TEM
• What are the disadvantages of an SEM?

  • expensive
  • damage to specimen
  • artefacts
  • dead specimen
  • black and white (false coloured)
  • training required
• What is the second type of light microscopy?

  Laser Scanning Confocal Microscopy
• how does laser scanning electron microscopy work?


  laser beams scan a specimen tagged with fluorescent dye, causing the dye to fluoresce (give off light) which is then focused onto a detector, generating an image
• Are the advantages of laser scanning confocal microscopy?

  • Images can be 3D
  • precise layers can be viewed at different depths
  • higher resolution than light microscopy
  • can see living specimens
  • can observe cell processes by tracking molecules
• Why does laser scanning confocal light microscopy have a higher resolution than light microscopy?

  a pinhole blocks any out of focus light
• What is a disadvantage of laser confocal light microscopy?

  more expensive and complex than light microscopy
• Prepare a dry mount…

  1. Section the sample
  2. place on slide
  3. cover with cover slip
• to prepare a wet mount…

  1. section sample and place on slide
  2. add a drop of water
  3. place cover slip on at a 45 degree angle
• to prepare a squash slide…

  1. make a wet mount
  2. press the cover slip down
• to make a smear slide…

  1. add a drop of sample to a slide
  2. use the edge of one slide to smear the sample
  3. place a cover slip on
• What are the 4 types of microscopy?

  • light microscopy
  • laser scanning confocal microscopy
  • scanning electron microscopy
  • transmission electron microscopy
• What is sectioning?

  Cutting a specimen into thin slices
• To calibrate a microscope…

  1. line up eyepiece graticule and stage micrometer
  2. work out the amount of eyepiece divisions in one stage division
  3. equate the value of 1 eyepiece division
  4. use the eyepiece divisions in magnification equations to find The actual size
• What is an eyepiece graticule?


  Transparent ruler fitted to the eyepiece with numbers but no units.
• What is a stage micrometer?

  a microscope slide with an accurate scale (it has units)
• You must re-calibrate the microscope after every different magnification you use.
• Why do we stain?


  the cytoplasm and other cell structures and tissues are often transparent. Stains increase contrast as different components of the cell take up stains to different degrees, allowing components to be seen more clearly
• What stains are used in light microscopy?


  Coloured dyes
• how do coloured dyes work in light microscopy?


  dyes absorb specific colours of light while reflecting others, making the structures which absorb them visible.
• Certain tissues absorb certain dyes depending on their chemical nature
• crystal violet and methylene blue are positively charged dyes which are attracted to negatively charged materials in the cytoplasm, staining cell components.
• Nigrosin and Congo red are negatively charged stains and are repelled by the negatively charged cytosol. They stay outside the cells, leaving them unstained against a stained background. This is a negative stain technique.
• What is a negative stain technique?


  when dyes stain the background and leave the cell unstained
• what is differential staining?


  staining with multiple dyes to ensure that different tissues in a specimen show up
• What is the gram stain technique used for?


  to separate bacteria into 2 groups, gram-positive bacteria and gram-negative bacteria.
• what type of stain technique is the gram stain technique?


  differential staining