Foundation in Biology
All living organisms are made up of cells, but cells are too small to be seen clearly with the naked eye. To investigate them in detail, scientists use microscopes as it allows us to make the sample bigger. Microscopy has allowed us to discover the structure of cells, understand their functions, and see how they differ across a wide variety of prokaryotic and eukaryotic organisms (plants, animals, fungi and protists).
Different microscopes produce different types of images.
A light microscope allows us to observe living cells and tissues, though only at limited magnification and resolution.
2.1.1 Cell structure - Microscopes
Course focusing knob – moves objective lens closer or further away from specimen by moving the stage
Fine focusing knob– moves objective lens in smaller steps to focus on particular parts of the specimen
The eyepiece lens produces a magnified ‘real image’ of the specimen (normally x10)
Magnification: The number of times greater that an image is than the actual object.
Objective lens are closest to the object and are responsible for both magnifying and increasing the resolution of the object.
Resolution: The ability to separate objects that are close together i.e. more detail.
When figuring out how much a sample has been magnified, you must take into consideration both the eyepiece magnification and the objective lens.
TOTAL MAGNIFICATION = EYEPIECE MAGNIFICATION X OBJECTIVE LENS MAGNIFICATION i.e. a x10 eyepiece lens is used with a x40 objective lens, therefore the total magnification is x400
When using a light microscope, the specimen often needs to be prepared in a way that makes it easier to observe. There are several common techniques you should be familiar with:
Dry mount: Useful for: hairs, pollen grains, insect parts, thin sections of plants.
Use a sharp blade to cut a thin slice (allows light to penetrate) or the whole specimen is placed on the slide with a cover slip on top.
Wet mount: Useful for: aquatic samples, living organisms (e.g. protists), and soft tissues that need support.
Sample is suspended in liquid (water/ immersion oil) to prevent dehydration/ distortion of tissue, cover slip is then lowered at an angle to prevent air bubbles
Squash slides: Useful for: soft samples, e.g. root tips when studying mitosis, to spread cells into a thin layer.
A wet mount is prepared, then gently press down (sometimes with tissue paper) on the cover slip or another microscope slide to squash the specimen.
Smear slides: Useful for: blood samples, to see individual cells clearly.:
A drop of liquid sample (i.e. blood) is placed on the slide using a pipette, then using the edge of another slide the liquid is smeared across the slides before adding a cover slip.
Squash slide showing root tip cells going though mitosis
When you look at a specimen under a microscope, the image is magnified – but the magnification alone doesn’t tell you the actual size of the structure. To measure accurately, you first need to calibrate the microscope.
Calibration is done by using two special tools:
Eyepiece graticule – a small transparent scale inside the eyepiece. It looks like a ruler, but it has no fixed units.
Stage micrometer – a tiny ruler on a microscope slide, with a scale where the divisions are known (usually 0.01 mm or 10 μm)
How to calibrate eyepiece graticule:
Place the stage micrometer on the stage and bring it in to focus.
Line up the stage micrometer with the scale on the eyepiece graticule
Work out how many divisions on the graticule match a known length on the micrometer i.e 2 divisions (2x10μm = 20μm) on the stage micrometer below lines up with 6 division on the eyepiece graticule
From this, calculate the value of one eyepiece division at that magnification. 1 eyepiece graticule division = 20/6 = 3.33μm
Repeat step 2-4 for each of the objective lens
Now, when you place your specimen slide under the microscope, you can use the calibrated graticule to measure the actual size of cells or structures.
i.e 100 divisions on the stage micrometer above lines up with 40 divisions on the eyepiece graticule: 1000/40 = 25μm
Answer: draw a ruler line from both sides of the vascular bundle down to the graticule scale as shown by the red lines in figure 1.
Count the number of division between the lines (13 divisions).
multiply 13 by the eyepiece graticule unit 25μm: 13 x 25 = 325μm
figure 1. vascular bundle seen under the microscope
Air bubble
Example exam practice: Calculate the diameter of the vascular bundle in figure 1. using the calculated eyepiece unit of 25μm
Staining samples
In your exam you may be asked to produce a scientific drawing from observations seen down the microscope. Some important rules that must be followed:
Your drawing and any label lines must be drawn using a really sharp pencil (not a pen).
Your drawing should take up at least half the page / space available.
Correct proportions i.e. different areas are the right size relative to each other, and that your drawing is a true likeness of the specimen that you are drawing. (you are not expected to be an artist)
label lines should be straight and drawn using a ruler (in pencil). Don’t let the label lines cross
Make sure the label lines touch the part you are labelling.
Always include a title and a scale bar
Include annotations about the structures/ features you have labelled.
