Using a 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
- 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
- Note that most of the colours seen in images taken using a light microscope are the result of added stains
- 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
- Methylene blue
Examples of Microscope Specimen Stains Table
Toluidine blue and phloroglucinol have been used to stain this tissue specimen taken from a leaf