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

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.