2.1.2 Using A Microscopes Flashcards
Why do we use Optical Microscopes
Many biological structures are too small to be see by the naked eye
Optical microscopes are an invaluable too for scientists as thy allow for tissues, cells and organelles to be 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 lens, the stage, the light source, the coarse and fine focus
Other tools used:
Forceps, scissors, scalpel, coverslip, slides, pipettes, standing solution
Method for Liquid Specimen
Add 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
Method for a Solid Specimen (simple)
Use scissors to cut a small sample of tissue
Peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide (using a sample or forceps)
-the tissue needs to be thin so that the light from the microscope can pass through
Apply a stain
Gently placed a coverslip on top and press down to remove any air bubbles
(Take care when using sharp objects and wear gloves to prevent the stain from dying your skin)
Method for a Solid Specimen (chemical)
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 specimen, a stain. Is applied and the specimen is mounted using a resin and a coverslip is applied
Method for a Solid Specimen (freezing)
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 stain
Gently place a coverslip on top and press down to remove any air bubbles
Why do you always start with the low power objective lens when using an optical microscope
It’s is easier to find what you are looking for in the field of view
This helps to prevent damage to the lens or the coverslip in case the stage has been raised too high
Preventing the Dehydration of Tissue
The thin layer of material can dry up rapidly
Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration
How to deal with Unclear or Blurry Images
Switch to the lower power objective lens and they using 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 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 2 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
Limitations
The size of cells or structures of tissues may appear inconsistent in different specimen slides
- Cell structures are 3-D and the different tissue samples will have been cut at different planes, resulting in these inconsistencies when viewed on a 2-D 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
Many tissues that are used in microscopy are naturally translucent (they let both light and electrons though 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 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 photo micro graphs (image taken using a light microscope) are not natural
-e.g. chloroplasts don’t need stains as they show up as green, which is their natural colour
Toluidine blue (stains cells blue) and phloroglucionol (stains cells red/pink) are common stain used
Staining for Electron Microscopy
When using 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 dye because the absorb electrons well
-osmium tetroxide and ruthenium tetroxide are example
Any colour present in electron micrograph so is not natural and it is also not a result of the staining
-coloured are added to the image using an image processing software
