Help! There is a comet in my computer! 40
Having a higher pixel depth brings another advantage. When we start recording images for
a comet experiment, we optimise the system to acquire images of good quality. We should
also not change the settings of image acquisition during recording of one experiment. The
problem is that when we are adjusting the settings of the system, we only see a few comets.
We do not know whether on other slides we will have comets with much brighter heads
than the ones we see. We also do not know whether some slides will have a lower level of
background signal than our current slide. As we have seen, our image must never be
saturated – it must always contain information on all variation in the intensity of the
background and the comet signal. To account for a possible lower background signal and a
higher comet signal in the comets that will be scored later, we adjust the camera settings at
the beginning of measurement so that we leave some unused grey values lower than the
current background values (Figure 23, marked in blue) and some unused grey values
higher than the current comet values (Figure 23, marked in orange). With a 10-bit camera
this is less problematic, since even if we leave about half of the range of available grey
values unused, we are still describing the signal with 500 grey values. Nevertheless, it is
possible to score comets with an 8-bit camera, as long as we are careful not to saturate the
image.
5.8 Binning
Some comet measurement systems offer an option of binning for image acquisition.
Binning is a setting of the camera – it specifies how light intensity is detected in the
camera detector. Without binning, each light-sensitive element in the camera records the
intensity of light, which is then represented as one pixel in the grabbed image. With
binning set to 2×2, the camera records the signal from 4 adjacent detector elements as one
pixel (Figure 24). With binning 3×3, the signal from nine detector elements is pooled
together.
Application of binning has two effects. The sensitivity of the camera increases – the
camera can detect a weaker light signal. On the other hand, the resolution of the camera
decreases – the grabbed image uses fewer pixels to record the same field of view, so we
see fewer details in the image, the comet ROI contains fewer pixels and the line graph
representing the comet profile is constructed from fewer intensity points.
Binning 2×2 is usually recommended for cameras with a high resolution. A camera with
full resolution 1300 × 1030 pixels records images with size 650 × 515 pixels at binning
2×2. A positive effect of binning for comet scoring is a higher sensitivity of the camera, so
that a weak fluorescence signal can be detected. In addition, at binning 2×2, the comet ROI
contains 4-times fewer pixels than in the full resolution image without binning (Figure 24),
so the computer can do the calculations of measured parameters faster. In practice, we
always make compromises between camera resolution and sensitivity – we need to detect
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