1.2.16 Practical: Using a Light Microscope

• 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

• In order to determine the size of an object being viewed, a microscope needs to be calibrated using the eyepiece graticule in combination with a slide known as a stage micrometer
• Most microscopes contain an internal ruler within the eyepiece known as the eyepiece graticule
  • The ruler is a series of vertical lines that are evenly spaced
  • The scale will measure differently depending on the objective lens magnification being used, i.e. 4x, 10x or 40x
  • There may be small differences between the precise magnification of different microscopes, which will also affect the measurements of the eyepiece graticule
• A stage micrometer is a slide with an accurate scale in micrometres (µm)
  • Most stage micrometers have a scale in which the smallest subdivisions measure 0.01 mm, or 10 μm
• Calibrating the graticule with the stage micrometer allows us to work out the actual length of each unit in the eyepiece graticule ruler (known as graticule divisions), and we can then use the graticule ruler to measure the object being viewed at a specific magnification with a specific microscope
• To calibrate the eyepiece graticule it is lined up with the stage micrometer:

The stage micrometer (upper scale) and the eyepiece graticule (lower scale) can be used together to work out the actual size of an object being viewed.

• In the diagram the stage micrometer has three lines that are visible, so there are two micrometer divisions within the field of view
• Each micrometer division corresponds to 40 divisions on the eyepiece graticule (between 10-50, and between 50-90)
• This means that one micrometer division (which measures 10 μm; see above) is equal to 40 graticule divisions
• Using this information we can calculate the actual length of one graticule division as follows

40 graticule divisions = 10 μm

1 graticule division = 10 ÷ 40

(we divide 40 by itself to give 1 graticule division on the left, so we divide 10 by the same factor on the right)

1 graticule division = 0.25 μm

• The specimen slide would be used to replace the stage micrometer and the eyepiece graticule would be used (at the same magnification) to measure the length of the object
• The number of graticule divisions spanned by the object can then be multiplied by the magnification factor to work out the size of the object:

graticule divisions x 0.25 = size of object (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