• 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

   

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

   

• Preparing a slide using a?solid specimen:
  • 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)
  • Some tissue samples need be treated with chemicals to kill/make the tissue rigid
  • Gently place a coverslip on top and press down to remove any air bubbles
  • A?stain?may be required to make the structures visible depending on the type of tissue being examined
  • Take care when using sharp objects and wear gloves to prevent the stain from dying your skin

   

• 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 incase 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 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

• The gas exchange surfaces of different organisms can be observed using microscopes
• They often appear very different in photomicrographs than they do in the diagrams found in textbooks
• It is important to be able to identify the gas exchange surface and the?key structures?present

Mammal Gas Exchange

• A section of stained lung tissue can be seen in the image below
• The?alveoli?are of different sizes and shapes
  • This is because they are no longer inflated as they would be in a living lung

   

• The?nuclei?are shown as dark dots
• Blood vessels?can found in between the alveoli
• Sometimes white blood cells are present in tissue samples

Image showing a section of stained lung tissue

Fish

• A section of stained fish gills taken from a dogfish are shown in the image below
• The?gill arch?resembles a backbone for the gills
• The different?filaments?are shown with many of the?lamellae?visible

Image showing a section of fish gills taken from a dogfish

Insect

• As insects are very small obtaining a clear image of their gas exchange system can be difficult
• Electron microscopes?can take clear images of the?spiracle?structures found on the surface of insects, like the one shown below

Image showing a spiracle found in the wall of a caterpillar

Dicotyledonous leaves

• Plants often provide good specimens for microscopy
• A section of stained tissue from a dicotyledonous leaf is shown in the image below
• The different layers of tissue and cell types are clear
  • Waxy cuticle
  • Epidermal layers
  • Palisade mesophyll layer
  • Spongy mesophyll layer

   

• The?stomata?are visible with the?guard cells?on either side

Image showing a section of stained leaf