• 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

   

• Use a?calibrated?graticule to take measurements of cells

Using a graticule

• An?eyepiece graticule?and?stage micrometer?are used to measure the size of the object when viewed under a microscope
• Each microscope can vary slightly so?needs to be calibrated?when used
• The calibration is done with a stage micrometer, this is a slide with a very accurate scale in?micrometres (μm)
• The eyepiece graticule is a disc placed in the eyepiece with 100 divisions, this has no scale
• To know what the divisions equal at each magnification the eyepiece graticule is calibrated to the stage micrometer at each magnification

• In the diagram, the stage micrometer has three lines each 1000 μm (1 mm) apart
• Each 1000 μm division has 40 eyepiece graticule divisions
• 40 graticule divisions = 1000 μm

1 graticule division = number of micrometres ÷ number of graticule division

• 1 graticule division = 1000?÷?40 = 25 μm this is the?magnification factor
• The specimen slide would be used to replace the stage micrometer and the eyepiece graticule at the same magnification would be used to measure the length of the object
• The number of graticule divisions can then be multiplied by the magnification factor:

graticule divisions x magnification factor = measurement (μm)

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