Practical 1: Using a Microscope
- Many biological structures are too small to be seen by the naked eye
- Optical (light) 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 (light) 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 (light) microscope are:
- The eyepiece lens
- The objective lenses
- The stage
- The light source
- The coarse and fine focus
- Forceps
- Scissors
- Scalpel
- Coverslip
- Slides
- Pipette
Image showing all the components of an optical (light) 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
- 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)
- Some tissue samples need be treated with chemicals to kill/make the tissue rigid
- A stain may be required to make the structures visible depending on the type of tissue being examined
- Gently place 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
- 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 incase the stage has been raised too high
- Do not move the stage down when moving to higher objective lens
- 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
- A graticule is a small disc that has an engraved scale. 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
Drawing cells
- To record the observations seen under the microscope (or from photomicrographs taken) a labelled biological drawing is often made
- Biological drawings are line pictures which show specific features that have been observed when the specimen was viewed
- There are a number of rules/conventions that are followed when making a biological drawing
Guidelines for microscope drawings
- The conventions are:
- The drawing must have a title
- The magnification under which the observations shown by the drawing are made must be recorded
- A sharp HB pencil should be used (and a good eraser!)
- Drawings should be on plain white paper
- Lines should be clear, single lines (no thick shading)
- 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 be kept to one side of the drawing (in parallel to the top of the page) and drawn with a ruler
- Drawings of cells are typically made when visualising cells at a higher magnification power, whereas 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 drawn from a low-power image of a transverse section of a root. There is no cell detail present.
Magnification calculations
- Magnification is how many times bigger the image of a specimen observed is in comparison to the actual (real-life) size of the specimen
- The magnification (M) of an object can be calculated if both the size of the image (I), and the actual size of the specimen (A), is known
An equation triangle for calculating magnification
Worked example
An image of an animal cell is 30 mm in size and it has been magnified by a factor of X 3000.What is the actual size of the cell?
To find the actual size of the cell:
Using the appropriate units
- The size of cells is typically measured using the micrometre (μm) scale, with cellular structures measured in either micrometers (μm) or nanometers (nm)
- When doing calculations all measurements must be in the same units. It is best to use the smallest unit of measurement shown in the question
- To convert units, multiply or divide depending if the units are increasing or decreasing
- Magnification does not have units
- There are 1000 nanometers (nm) in a micrometre (µm)
- There are 1000 micrometres (µm) in a millimetre (mm)
- There are 1000 millimetres (mm) in a metre (m)
Using a scale bar
- A scale bar is a straight line on the drawing or micrograph that represents the actual size before the image was enlarged
- If the calculation required includes a scale bar on the micrograph or drawing then follow these steps:
- Use a ruler to measure the length of the scale bar in millimetres
- Convert this measurement into the same units as the number on the scale bar
- Insert these numbers into the magnification formula above (note: the size of the image is the measured length of the scale bar and the actual size is the number on the scale bar)
Worked example
Calculate the magnification of the transverse section of the leaf blade.
Transverse section of the leaf blade
Step 1: Use a ruler to measure the length of the scale bar in millimetres
Using a ruler the length of the scale bar is equal to 20 mm
Step 2: Convert this measurement into the same units as the number on the scale bar
The units on the scale bar are µm, remember that 1mm = 1000 µm
therefore 20 mm = 20 x 1000 = 20 000 µm
Step 3: Insert these numbers into the magnification formula
Note: the size of the image is the measured length of the scale bar and the actual size is the number on the scale bar
therefore Magnification = x 100
Exam Tip
Before doing any calculations make sure that all the measurements have the same units.When doing the calculations it is easier to write the formula, then rearrange it, before you add any measurements, as this helps avoid any possible errors.Note that when you do calculations using a scale bar, the number on the scale bar is informing you how many mm/µm or nm the line actually represents (e.g. if the scale bar has 20 nm above it and the line is 10 mm, then every 10 mm on the diagram is actually 20 nm).