Measuring the size of your sample, or features within your sample, by using microscopy can be done in different ways but all depend on knowing the extent of magnification in one way or another. Magnification was described above and is controlled by the objective lens in the microscope. This is the lens nearest to the sample. The higher the magnification, the smaller the field of view will be.

Describing an image as showing a given magnification only applies to a fixed display size. If an image is enlarged, then so is its magnification. When images were captured onto a fixed-sized photographic film negative, it was clear that the negative showed a certain magnification such as 200x or 50,000x and that any increase in size when an image was printed could easily be calculated and used to increase the magnification factor. Magnification for digital images is usually calibrated in the microscope software. If it hasn’t been, or to check the calibration, capture an image of a stage micrometer. You can then determine the pixel size by looking at how many pixels in the image cover a measured distance on the micrometer. In a light microscope, you can also use a haemacytometer for this if you don’t have a stage micrometer.

Other more effective ways to represent the extent of magnification, and therefore the size of features in the image, regardless of display size include:

• describing the horizontal field width (HFW). HFW means the width of the area captured in the image. You can then calculate the proportion of this that is taken up by your feature of interest. Note: micrometre is abbreviated as µm and nanometre as nm.
  For example, if the HFW is 25µm, and your feature of interest takes up 20% of the width of the image, then 25µm x 20% = 5µm. So, your feature is 5µm wide.
• use of a scale bar. These are often added by the instrument software and provide another easy way to calculate the size of different components in the image. When doing this, all measurements must be done on the image at the same dimensions – you can’t zoom in further if you have already made one of the measurements.

First measure the length of the scale bar (on screen or on a printed page). Say this comes to 11 mm. Then, measure the width of your feature of interest; in this case say it is the distance between the long ridges, which measures about 17.5 mm (on the screen or printed page). Next, note the length that the scale bar represents, which is 1 µm (the number next to the scale bar).

Divide the size of the feature by the measured length of the scale bar and multiply by the length represented by the scale bar. This will give you the real width of the feature: (17.5/11) x 1µm = 1.59µm.