• Many biological structures are too small to be seen by the naked eye
• Optical microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and studied
• For example, the movement of chromosomes during mitosis can be observed using a microscope

How optical microscopes work

• Light is directed through the thin layer of biological material that is supported on a glass slide
• This light is focused through several lenses so that an image is visible through the eyepiece
• The magnifying power of the microscope can be increased by rotating the higher power objective lens into place

Apparatus

• The key components of an optical microscope are:
  • The eyepiece lens
  • The objective lenses
  • The stage
  • The light source
  • The coarse and fine focus

   

• Other tools used:
  • Forceps
  • Scissors
  • Scalpel
  • Coverslip
  • Slides
  • Pipette
  • Staining solution

   

Image showing all the components of an optical microscope

Method

• Preparing a slide using a?liquid specimen:
  • Add a few drops of the sample to the slide using a pipette
  • Cover the liquid/smear with a coverslip and gently press down to?remove air bubbles
  • Wear gloves?to ensure there is no cross-contamination of foreign cells

   

• Methods of preparing a microscope slide using a?solid specimen:
  • Take care when using sharp objects and wear gloves to prevent the stain from dying your skin
  • Use scissors to cut a small sample of the tissue
  • Peel away or cut a?very thin layer?of cells from the tissue sample to be placed on the slide (using a scalpel or forceps)
    • The tissue needs to be thin so that the?light?from the microscope can pass through

     

  • Apply a stain
  • Gently place a coverslip on top and press down to remove any air bubbles

   

Or

• • Some tissue samples need to be treated with chemicals to kill/make the tissue rigid
  • This involves?fixing the specimen?using formaldehyde (preservative), dehydrating it using a series of ethanol solutions, impregnating it in paraffin/resin for support then cutting thin slices from the specimen using a?microtome
  • The paraffin is removed from the slices/specimen, a stain is applied and the specimen is mounted using a resin and a coverslip is applied

   

Or

• • Freeze?the specimen in carbon dioxide or liquid nitrogen
  • Cut the specimen into thin slices using a?cryostat
  • Place the specimen on the slide and add a stain
  • Gently place a coverslip on top and press down to remove any air bubbles

   

• When using an optical microscope always?start with the low power objective lens:
  • It is?easier to find?what you are looking for in the field of view
  • This helps to?prevent damage?to the lens or coverslip in case the stage has been raised too high

   

• Preventing the dehydration of tissue:
  • The thin layers of material placed on slides can?dry up rapidly
  • Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration

   

• Unclear or blurry images:
  • Switch to the lower power objective lens and try using the?coarse focus?to get a clearer image
  • Consider whether the specimen sample is?thin enough?for light to pass through to see the structures clearly
  • There could be?cross-contamination?with foreign cells or bodies

   

• Using a graticule to take measurements of cells:
  • A?graticule?is a small disc that has an?engraved ruler
  • It can be placed into the eyepiece of a microscope to act as a ruler in the field of view
  • As a graticule has no fixed units it must be?calibrated?for the objective lens that is in use. This is done by using a scale engraved on a microscope slide (a stage micrometer)
  • By using the two scales together the number of micrometers each graticule unit is worth can be worked out
  • After this is known the graticule can be used as a?ruler?in the field of view

   

The stage micrometer scale is used to find out how many micrometers each graticule unit represents

Limitations

• The size of cells or structures of tissues may appear inconsistent in different specimen slides
  • Cell structures are 3D and the different tissue samples will have been?cut at different planes?resulting in this inconsistencies when viewed on a 2D slide

   

• Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that can not be seen
• The treatment of specimens when preparing slides could alter the structure of cells

• Many tissues that are used in microscopy are naturally?transparent, they let both light and electrons pass through them
• This makes it very difficult to see any detail in the tissue when using a microscope
• Stains are often used to make the tissue coloured/visible

Staining for light microscopy

• Coloured dyes are used when staining specimens
• The dyes used?absorb specific colours of light while reflecting others; this makes the structures within the specimen that have absorbed the dye visible
• Certain tissues absorb certain dyes, which dye they absorb depends on their chemical nature
• Specimens or sections are sometimes?stained with multiple dyes?to ensure the different tissues within the specimen show up - this is known as?differential staining
• It is important to remember that most of the?colours?seen in photomicrographs (image taken using a light microscope) are?not natural
  • Chloroplasts don't need stains as they show up green, which is their natural colour

   

• Toluidine blue and phloroglucinol?are common stains used
  • Toluidine blue turns cells blue
  • Phloroglucinol turns cells red/pink

   

Toluidine blue and phloroglucinol have been used to stain this tissue specimen taken from a leaf

Staining for electron microscopy

• When using Transmission electron microscopes (TEMs) the specimen must be stained in order to?absorb the electrons
• Unlike light, electrons have?no colour
  • The dyes used for staining cause the tissues to show up?black or different shades of grey

   

• Heavy-metal compounds?are commonly used as dyes because they absorb electrons well
  • Osmium tetroxide and ruthenium tetroxide are examples

   

• Any of the colour present in electron micrographs is not natural and it is also not a result of the staining
• Colours are added to the image?using an image-processing software

The internal structure of the mitochondrion can be seen using a TEM and staining

A spiracle found on the exoskeleton of an insect. No colours have been added to this image using image-processing software.