ChemoMetec NC-100 - Manual (Page 33)

4 Measurement tab
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axis is signal intensity on a scale from 0 to 255. The unit of the y -axis is the number of
stained nuclei or objects.
Figure 19 The object intensity histogram.
Variation of the background level of the image gives rise to a number of signals of low
intensity. These signals are found to the far left of the object intensity hi stogram,
generally the intensities of the background signals are below 20.
The stained nuclei give rise to higher signal intensities, which is illustrated in figure 19, as
the rise in the number of signals to the right of the background signals. There is often an
increase in the number of signals at the maximum intensity (255). These signals originate
from stained nuclei having fluorescence intensities, which exceed the dynamic range of the
detection system. These nuclei are also included in the counts.
Since there can be fluorescent objects with intensities which are only slightly higher in
intensity than features in the background it is necessary to determine a limit, which
discriminates between signals from a nuclei and signals from objects in the background. At
the signal intensity of 50 the discriminator is placed. The discriminator defines that any
object with signal intensity above 50 is included in the cell count, while signals of
intensities below 50 are regarded as background signals.
Due to the high signal to noise ratio of the NucleoCounter, illustrated by the low
frequencies of signals of intensities between 20 and 60, the position of this discriminator
is not critical. When measuring samples of similar origin (mammalian cells) the
fluorescence signals from all nuclei will have similar intensities since the DNA content of
nuclei is similar. Thus the discriminator does not have to be adjusted individually for each
sample.
4.9 Save image
The image of the NucleoCassette chamber can be saved for documentation purposes. How
to save the image is explained below.