OCR A Level Biology:复习笔记2.1.2 Using a Microscope

Preparation of Microscope Slides

  • 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

Exam Tip

Remember the importance of calibration when using a graticule. If it is not calibrated then the measurements taken will be completely arbitrary!

Staining in Light Microscopy

  • 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.

 

 

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