Help! There is a comet in my computer! 36
Uneven illumination of the field of view cannot be completely eliminated even in an
optimally aligned microscope. Hence, it is good practice to place all measured comets in
the same position on the image during image acquisition, close to the centre of the field of
view, where shading is least severe in a well-aligned microscope.
A weak shading effect is partially corrected during calculation of the comet parameters.
You will remember that to create a comet profile, we calculated the light intensities for
each individual column of pixels, taking always into account the local background in each
respective pixel column (Figure 10, Table 1). In this way, a small gradient of background
grey values running from left to right (along the x-axis) can be corrected. However, severe
shading and shading in the direction from top to bottom of the image (along the y-axis) is
not accounted for and can lead to erroneous results.
When DNA-bound ethidium bromide is continuously exposed to strong excitation light,
the intensity of the emitted signal gradually decreases. Excitation of the comets
surrounding the current field of view can be minimised by closing of the diaphragm in the
excitation light path. Note, however, that some shading can occur if the edge of the
diaphragm aperture is too close to the edge of the field recorded by the camera. So we have
to make sure that the diaphragm is not closed too tightly.
5.6 How can I detect shading in my image?
Shading cannot be detected by just looking at an image – our eyes cannot detect gradual
changes in the shades of grey in the very dark background. One indicator of shading can be
seen in the comet profile. In severe cases of shading (and where the gradient of the
background signal runs from left to right), the background line of the comet profile is
sloping (without shading, it is horizontal).
To detect shading in an image, we will use the “Levels” and “Threshold” tools of
Photoshop (see chapter How can I evaluate the quality of my image?, page 32 for a
description of these tools and explanation about import of a comet image to Photoshop).
We will analyse an image with an acceptable level of shading (Figures 22A, 22B) and an
image with a severe shading effect (Figures 22C, 22D).
One of the indicators of shading is a wide peak of background grey values in the histogram
of image grey values (Figures 22A, 22C; Photoshop: from main menu Image /
Adjust(ments) / Levels).
The best approach for visualisation of shading is application of the “Threshold” tool in
Photoshop (from the main menu Image / Adjust(ments) / Threshold). If we then drag the
slider for threshold grey values under the histogram, we can observe the distribution of
grey values in the background. In an image with an acceptable level of shading, the whole
background appears even within the range of a few grey values (Figure 22B: 1-5). This
image has some shading – background in the left side of the image is slightly darker than
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