3.1.5 Microscopy

• 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
  • Light is directed through a 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

Preparation of microscope slides

• 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 that may be used
  • Forceps
  • Scissors
  • Scalpel
  • Coverslip
  • Slides
  • Pipette
  • Staining solution

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 or a scalpel to cut a small sample of the tissue
  • Use forceps to peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide 
    • The tissue needs to be thin so that the light from the microscope can pass through
  • Apply a stain to make cells more visible
  • Gently place a coverslip on top and press down to remove any air bubbles

• Some tissue samples need to be treated with chemicals to kill cells or make the tissue rigid
  • This involves fixing the specimen using the preservative formaldehyde, dehydrating it using a series of ethanol solutions, impregnating it with paraffin or resin for support and then cutting thin slices from the specimen
  • The paraffin is removed from the slices and a stain is applied before the specimen is mounted and a coverslip is applied

Slide Preparation Table

Using a microscope

• 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

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 cannot be seen
• The treatment of specimens when preparing slides could alter the structure of cells

Staining in light microscopy 

• Specimens to be viewed under a microscope sometimes need to be stained, as the cytoplasm and other cell structures may be transparent or difficult to distinguish
  • Note that most of the colours seen in images taken using a light microscope are the result of added stains
    • Chloroplasts are the exception to this; they show up green, which is their natural colour
• The type of stain used is dependent on what type of specimen is being prepared and what the researcher wants to observe within the specimen
  • Different molecules absorb different dyes depending on their chemical nature
• Specimens or sections are sometimes stained with multiple dyes to ensure that several different tissues within the specimen show up; this is known as differential staining
• Some common stains include
  • Methylene blue
    • Stains animal cell nuclei blue
  • Iodine
    • Stains starch-containing material in plant cells blue-black
  • Toluidine blue 
    • Stains tissues that contain DNA and RNA blue
  • Phloroglucinol
    • Stains a chemical called lignin found in some plant cells red/pink

Examples of Microscope Specimen Stains Table

Toluidine blue and phloroglucinol have been used to stain this tissue specimen taken from a leaf

• To record the observations seen under the microscope, or from photomicrographs taken, a labelled biological drawing is often made
  • Biological drawings are line drawings that show specific features that have been observed when the specimen was viewed
• There are a number of rules or conventions that are followed when making a biological drawing
  • The drawing must have a title
  • The magnification under which the observations shown by the drawing are made must be recorded
  • A sharp pencil should be used 
  • Drawings should be on plain white paper
  • Lines should be clear, single lines with no sketching
  • No shading
  • The drawing should take up as much of the space on the page as possible
  • Well-defined structures should be drawn
  • The drawing should be made with proper proportions
  • Label lines should not cross or have arrowheads and should connect directly to the part of the drawing being labelled
  • Label lines should ideally be kept to one side of the drawing in parallel to the top of the page, and should be drawn with a ruler
  • Only visible structures should be drawn; not structures that the viewer thinks they should be able to see!

• Drawings of cells are typically made when visualizing cells at a higher magnification power
• Plan drawings are typically made of tissues viewed under lower magnifications
  • Individual cells are never drawn in a plan diagram

An example of a tissue plan diagram drawn from a low-power image of a transverse section of a root. Note that there is no cell detail present.

An example of a cellular drawing taken from a high-power image of phloem tissue