Help! There is a comet in my computer! 14
3.5 Measurement of light intensity parameters
Several parameters measured in the comet assay describe the intensity of light emitted
from the comet slide – they are called the light intensity parameters. Their measurement
is based on information contained in the grey values of an image and they describe the
quantity and distribution of DNA in a comet. The very good news is that we only need to
measure the relative amount of DNA (e.g. light intensity in relative units, the percentage
of DNA in the comet tail) and not the absolute amount of DNA (in picograms or the
number of basepairs).
3.5.1 Integrated light intensity
Let us first take a look at the integrated light intensity – the sum of all light intensities in
a region of interest (ROI; the head or the tail). This parameter is shortly called the
integrated intensity or the total intensity. Because there is a linear relationship between
the intensity of light emitted from a comet and the grey values in an image, the integrated
light intensity should be equal to the sum of grey values for all pixels in a ROI.
To measure the integrated light intensity for the head of the model comet image, we need
to know the grey values of all pixels in the head, and then add the grey values together
(Figure 8). The result is 23903. Calculated with the same principle, the integrated light
intensity of the tail is 17371.
However, these results are not accurate. The problem is that the background, the part of
the image where there is no comet, is not completely black. In other words, the grey values
of the background are not 0; in the model comet image they range from 41 to 51. The
camera is detecting a weak signal in the background, and we will see that the background
signal is as important for our measurement as the comet signal.
Several factors may contribute to the background signal. Ethidium bromide around the
comets may emit light. The camera always has some electronic noise (it “randomly”
records grey values higher than 0 when there is no light). The mercury lamp may flicker a
little, all parts of the equipment are plugged in the electricity network, which does not have
a completely stable voltage, and this adds some more noise to the image. As we will learn
later, the background on the recorded image should never be completely black anyway (see
chapter Image saturation, page 30).
So we have to figure out how to take the background signal into account in our
measurement. In the parts of the image where there is no comet, we detected some light
intensity (background intensity). Physics teaches us that light intensities are additive. We
can thus deduce that in the parts of the image where there is the comet, the signal that we
recorded was the light intensity of the background plus the intensity of light emitted from
DNA-bound ethidium bromide. Hence, we need to subtract the background light
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