Techniques

• Microscopy:

  microscopes are used to investigate structures too small to be seen by the naked eye. The detail depends on magnification and resolution

  • magnification can be defined as the number of times bigger the image appears compared to its actual size
  • resolution is the minimum distance between 2 objects/points at which they can be seen separately (how detailed the image is). The higher the resolution, the smaller the organelles that can be seen
• Light (optical) microscopes:

  light microscopes use light waves and lenses to magnify an object. Light has a relatively long wavelength and so the resolution is low. Specimens must be thin enough (1 cell thick) to allow light to pass through. The maximum effective magnification is 1500x
• Evaluation of light microscopes:

  Pros - can view living or dead specimens and also get colour images. Relatively straightforward sample preparation and no vacuum needed

  Cons - low resolution; cannot see small organelles (ribosomes)
• Transmission electron microscope (TEM):

  these work on the principle that the beam of electrons is transmitted through the specimen. The denser part of the specimen absorbs more electrons, so the image looks darker. The specimen must be very thin and stained using electron-dense substances. Higher magnification than optical
• Evaluation of TEMs:

  Pros - highest resolution; can see very small organelles (internal structures of organelles - cristae in the mitochondria). This is because electrons have a shorter wavelength compared to light

  Cons - only view dead specimens and obtain black and white images. More complex specimen preparation - has to be done in a vacuum and cut more thinly than optical. Artefacts can appear on the specimen (dust)
• Scanning electron microscope (SEM):

  SEM record the electrons that are reflected off the surface of the object rather than passing through it. This produces a 3D image of external structures of a cell/structure. Higher magnification than optical
• Evaluation of SEMs

  Pros - higher resolution than optical - can see external structures in high detail. Gives a 3D image. Less complex preparation of specimens - thicker specimens can be used

  Cons - can only view dead specimens and obtain black and white images. Cannot see internal structures of cells (lower resolution than TEM). Specimens must be viewed in a vacuum
• Preparing a temporary mount for an optical microscope:

  Depending on the sample you are observing, you can prepare a sample in different ways. The following steps describe how you could make a temporary mount;

  • pipette a drop of water onto the centre of a slide
  • use forceps to place a thin section of the specimen on top of the water drop. The specimen should be 1 cell thick (light can pass through)
  • add a drop of a stain (iodine in potassium iodide). Stain will show the internal structures of the cells
  • use a mounting needle to lower a coverslip onto the specimen (avoiding any air bubbles)
• Drawing cells:

  • there should be no shading
  • drawings should have sharp, clear lines with no sketching
  • label lines should not cross and should be drawn with a ruler
  • don't draw in 3D; gave a 2D, flat view
  • there should be no pen used - not even for labelling
  • drawings should cover the majority of the space provided
  • scale bar or magnification should be given where appropriate
• Magnification units:

  the two units commonly used to describe microscopic objects are the micrometre (μm) and the nanometre (nm)

  One thousandth of a millimetre is known as a micrometre (μm)

  • 1000 μm = 1 mm
  • 1 mm = 1x10^-3 μm
• Magnification units:

  • the micrometre is used to measure cells and organelles. The average animal cell is 30 to 50 μm across. The nucleus has a diameter of about 10 μm. Plant cells are often larger and can be 150 μm across
  • Small organelles, such as ribosomes, and molecules are measured in nanometres (nm)
  • as a rough guide, a light microscope reveals structures that can be measured in micrometres, but you need an electron microscope to see objects measured in nanometres
• Magnification units:

  1 nanometre (nm) is one thousandth of a micrometre (μm)

  • 1 nm = 0.001 μm
  • so there are 1000 nanometres in a micrometre
  • therefore there is 1x10^6 or 1,000,000 nm in 1 mm

  Photographs taken through a light microscope (photomicrographs) or an electron microscope (electron micrographs) normally show how many times the image on paper is magnified. This is done by showing a magnification (e.g. x400), or by using a scale bar with the actual length it represents
• Magnification:

  magnification can be calculate using;

  magnification = image size ÷ actual size

  • the actual size is the true size of the object in real life. This is measured in mm or nm for cells or organelles
  • the image size is the size of the diagram or drawing. You may have to measure this with a ruler
• Measuring cells and organelles using eyepiece graticules:

  • an eyepiece graticule is needed to measure. This is a piece of equipment that goes in the eyepiece lens of an optical microscope. It allows you to look down at your sample against a scale/ruler
• Problems with eyepiece graticules:

  • light microscopes can have their magnification altered all the time, therefore the scale we can see is not a set length. When using a ruler during normal work the length will never change. One small division always represents a millimetre. This is not true for the eyepiece graticule scale as the magnification changes all the time
  • we need to calibrate it to be able to use it. Every time we adjust the magnification of the microscope we need to recalibrate the microscope
• Calibrating eyepiece graticule:

  • in order to calibrate the eyepiece graticule scale, we need to use a stage micrometre, which is a slide containing a ruler of set divisions. For this stage micrometre each division represents 0.01mm
  • each division of eyepiece graticule (μm) = length of stage micrometre (μm) ÷ number of eyepiece divisions