Comet Labs WN591 Manual de usuario descargar pdf (Pagina 38)

Help! There is a comet in my computer! 35

5.5 Shading

Our measurement was based on the assumption that a linear relationship exists between the

amount of DNA on the slide and the light intensity that the camera detected. One of the

factors that can endanger this relationship is shading of the image. We talk about shading

when there is a gradient in the intensity of the background signal across the image. We

usually get this effect when the whole field of view under a microscope is not evenly

illuminated during image acquisition.

The intensity of light emitted by DNA-bound ethidium bromide depends upon the intensity

of the green excitation light. Because we only measure the relative amount of DNA, the

actual excitation intensity is not so important, as long as we have a strong enough signal

for detection. However, the whole field of view has to be evenly illuminated, so that all

comets and all parts of one comet on an image receive the same intensity of excitation

light. We need to optimally align all components of the optical system. In particular,

careful centring of the mercury lamp is essential.

When shading occurs, the detected intensity of light emitted from a comet depends on the

position of this comet in the field of view, and consequently in the grabbed image. In

addition, if the field of view is not evenly illuminated, we can encounter strong gradients

of the background signal (Figure 21). The combined effects of shading compromise the

linearity and the accuracy of measurement, hence images with a strong shading effect

should not be measured.

Figure 21. Effects of uneven illumination of the field of view. In an evenly illuminated field of view

under a microscope, the background in the image is even and the recorded grey values of the

comet (shown as a filled circle) are the same for all positions of the comet on the image (A). On the

other hand, in an evenly illuminated field of view, the background in the image shows a gradient of

grey values and the recorded grey values of the comet are different for different positions of the

object on the image.

even illumination of the slide

uneven illumination of the slide (shading)

even illumination of the slide

uneven illumination of the slide (shading)

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Comet Labs WN591 Manual de usuario Pagina 38