Comet Labs WN591 Bedienungsanleitung PDF herunterladen (Seite 20)

Help! There is a comet in my computer! 17

However, we will now not average the grey values for the whole background region – we

will instead calculate the average background grey value for each column of pixels

separately. Why we need to do it this way will become clear later – it is to minimise the

measurement error (see chapter Shading, page 35).

Let us take the pixel column number eight (outlined in dark blue in Figure 8) as an

example. For this column, the sum of grey values for the 11 pixels in the head is 2443. In

this column, the sum of grey values for the 4 pixels in the background is 169, so the

average grey value of background pixels is 42.25. Therefore, we need to subtract 42.25

from each pixel in the head column. But because there are 11 pixels in the head column,

we can subtract 11 × 42.25 = 464.75 from the sum of grey values of the column: 2443 –

464.75 = 1978.25. This is the background-corrected integrated light intensity for pixel

column number eight in the head. After we repeat this procedure for all pixel columns in

the comet region, we can present the results in a table (Table 1). The background-

corrected comet intensity is our best estimate of the actual intensity of the light

emitted from the slide and is linearly proportional to the amount of DNA.

Now that we have a good estimate of the true intensities recorded by our camera, we can

calculate the total intensity of the head and the tail, by adding up intensities for all pixel

columns in each region. For the model comet image, the total intensity of the head is

18873, and the total intensity of the tail 10920. The total intensity of the whole comet is

thus 29793, the sum of the head and the tail intensities.

3.5.2 Percentage of DNA in the tail

One of the widely used comet parameters is the percentage of DNA in the tail. It is

calculated as the ratio between the total intensity of the tail and the total intensity of the

comet (head and tail together). For the model image of the comet, the percentage of DNA

in the tail is 10920 / 29793 = 0.37 = 37%.

To illustrate how important it is to eliminate sources of methodological error such as the

background signal, let us calculate the percentage of DNA in the tail without background

correction: 17371 / 41274 = 42%.

3.5.3 Tail extent moment

We can determine another comet parameter - the tail extent moment, defined as the

product of the tail length (also called the tail extent) and the percentage of DNA in the tail.

We already know the tail length for our model comet image, so we can now calculate the

tail extent moment: 22.4 µm × 37% = 8.3 µm. Note that the tail extent moment is

measured in absolute units - micrometers.

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Comet Labs WN591 Bedienungsanleitung Seite 20