Using a microscope

• Many biological structures are too small to be seen by the naked eye
• Optical microscopes


  An invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and studied
• How optical microscopes work

  1. Light is directed through the thin layer of biological material that is supported on a glass slide

  2. This light is focused through several lenses so that an image is visible through the eyepiece

  3. The magnifying power of the microscope can be increased by rotating the higher power objective lens into place
• Key components of an optical microscope

  • Eyepiece lens
  • Objective lenses
  • Stage
  • Light source
  • Coarse and fine focus
• Other tools used


  • Forceps
  • Scissors
  • Scalpel
  • Coverslip
  • Slides
  • Pipette
  • Staining solution
• Preparing a slide using a liquid specimen


  1. Add a few drops of the sample to the slide using a pipette

  2. Cover the liquid/smear with a coverslip and gently press down to remove air bubbles

  3. Wear gloves to ensure there is no cross-contamination of foreign cells
• Methods of preparing a microscope slide using a solid specimen

  1. Take care when using sharp objects and wear gloves to prevent the stain from dying your skin

  2. Use scissors to cut a small sample of the tissue

  3. 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)

  4. The tissue needs to be thin so that the light from the microscope can pass through

  5. Apply a stain

  6. Gently place a coverslip on top and press down to remove any air bubbles

  7. Some tissue samples need to be treated with chemicals to kill/make the tissue rigid
• Preparing a microscope slide using a solid specimen - chemical treatment

  1. Fix the specimen using formaldehyde (preservative)

  2. Dehydrate it using a series of ethanol solutions

  3. Impregnate it in paraffin/resin for support

  4. Cut thin slices from the specimen using a microtome

  5. Remove the paraffin from the slices/specimen

  6. Apply a stain

  7. Mount the specimen using a resin and apply a coverslip
• Preparing a microscope slide using a solid specimen - freezing

  1. Freeze the specimen in carbon dioxide or liquid nitrogen

  2. Cut the specimen into thin slices using a cryostat

  3. Place the specimen on the slide and add a stain

  4. 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
• Reasons for starting with 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

  Add a drop of water to the specimen (beneath the coverslip) to prevent the cells from being damaged by dehydration
• Dealing with unclear or blurry images

  1. Switch to the lower power objective lens

  2. Use the coarse focus to get a clearer image

  3. 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


  1. A graticule is a small disc that has an engraved ruler

  2. It can be placed into the eyepiece of a microscope to act as a ruler in the field of view

  3. The graticule must be calibrated for the objective lens that is in use using a stage micrometer

  4. The number of micrometers each graticule unit is worth can then be worked out

  5. The graticule can then be used as a ruler in the field of view
• The size of cells or structures of tissues may appear inconsistent in different specimen slides
• Reason for inconsistent cell/tissue size

  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
• Staining for light microscopy

  • Coloured dyes are used to make naturally transparent tissues visible
  • The dyes absorb specific colours of light while reflecting others, making the stained structures visible
  • Certain tissues absorb certain dyes, depending on their chemical nature
  • Specimens are sometimes stained with multiple dyes for differential staining
• Most of the colours seen in photomicrographs (image taken using a light microscope) are not natural
• Exceptions to staining


  Chloroplasts don't need stains as they show up green, which is their natural colour
• Common stains used


  • Toluidine blue
  • Phloroglucinol
• Staining for electron microscopy

  • The specimen must be stained in order to absorb the electrons
  • Unlike light, electrons have no colour
  • The dyes used 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
• Any colour present in electron micrographs is not natural and is added using image-processing software