User manual

Help! There is a comet in my computer! 45
6 Concluding remarks
I said “linear relationship” so many times that I would really like to avoid it in my
concluding remarks. But, here it comes again, because there really is only one rule for
measurement of comet parameters: Do not violate the linear relationship between the
amount of DNA and the grey values on the image of a comet (Figure 27). Once we have
a microscope, a good camera and a reliable software package, we have to make sure that
we are recording images of good quality. So for each experiment, we have to optimise
the settings of the microscope, the camera and any other settings that affect image quality.
We also have to select the right geometric calibration (image scaling factor). Only then can
we start to click.
When I started writing, I had no idea that I will need to explain so many things. I hope that
after you went through all the trouble of reading this lengthy paper, your understanding of
what your computer does to your comets has increased from grey value 0 (a completely
incomprehensible black box) to at least grey value 113 (a slightly mysterious grey box).
Figure 27. Measurement of the comet parameters: an overview.
amount of DNA on
a microscope slide
amount of EtBr
bound to DNA
intensity of
emitted light
linear
relationship
linear
relationship
linear
relationship
linear
relationship
stoichiometric binding
even illumination of the field of view
linear camera response
good image contrast
no saturated pixels
no shading
grey value
on an image
amount of DNA on
a microscope slide
amount of EtBr
bound to DNA
intensity of
emitted light
linear
relationship
linear
relationship
linear
relationship
linear
relationship
stoichiometric binding
even illumination of the field of view
linear camera response
good image contrast
no saturated pixels
no shading
grey value
on an image