Digital camera images and What can you see
jjmirujo at unav.es
Wed Mar 14 12:33:42 EST 2001
Peter Pediaditakis wrote:
> > While image analysis will provide an accurate measure of
> > density in the film, usually this is not the critical factor
> > in quantitation. If you run an internal standard, for
> > example several dilutions of a radioactive sample, and plot
> > O.D. vs radiactivity, you usually will find a linear
> > relation in a range. Below or above this range the
> > proportion does not apply. Moreover, in many cases the
> > extrapolated line does not cross the x=0, y=0 origin, so
> > doubling the amount of sample does not result in doubling
> > the measured O.D.
> First of all, unless nothing is there, you would expect the calibration
> line to not run through the (0,0) point. Second of all, usinfg the OD
> is a mistake. One pixel does not make a band. In order to properly
> extrapolate, you must find the integrated optical density (IOD) over the
> range of pixels that constitutes your band.
> > There may be many explanations for this
> > behaviour and this is the reason that internal standards
> > must be present. In fact, is the user, not the software, who
> > by chosing a variety of user-selectable parameters that
> > affect the quantitation (background subtraction, band
> > intensity, band contour...) takes the responsibility of the
> > result.
> The only user selectable parameter that is of importance is the contrast
> used during scanning. Background subtraction should happen to all pixels
> on the scan uniformly. Band intensity is what one is trying to find.
> Band contour, I have no idea what that is maybe you could elaborate.
There are different methods of removing background, and this greatly affects
the quantitation. Background subtraction can be applied either to the image
as a whole (as you suggest) or on individual lanes. A method called rolling
disk background subtraction is used to calculate the density that is removed
in each lane (the smaller the disk, the more density will be detected as
background). This method is appropriate when the background of the lane is
not uniform or is clearly different from gel background. Even if the
background is subtracted to the whole gel, artefacts arise if less or more
than correct value is removed.
On the other hand, gel analysis software allows the user to define bands in
several ways. It is suggested that you try out different parameter settings
in order to determine which are best for your gel (sensitivity, minimum
density, noise filter, shoulder sensitivity...) If the bands on the gel are
irregularly shaped, it is possible to use the contour function (contouring
will draw a lasso around a band and use the O.D. of all the pixel within
the boundary to determine band quantitation).
In any case, internal standards are very useful to decide the best method in
Juan J. Martinez Irujo
Depart de Bioquimica
Universidad de Navarra
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