Re: H DAB Color Deconvolution vectors

Posted by Gabriel Landini on Nov 02, 2005; 3:48pm
URL: http://imagej.273.s1.nabble.com/H-DAB-Color-Deconvolution-vectors-tp3704334p3704338.html

On Wednesday 02 November 2005 14:53, No Name wrote:
> I know that I need to measure our actual chromagens separately and then
> put these (normalized values) into the user values boxes or add them to
> the code. This is also noted by Landini on his page. I am in the process
> of doing this now since I know that my chromagens are pretty different
> from the default vectors.

Yes, that is the best: stain the images with only 1 chromogen at a time and
extract the vectors that way.

> The interesting thing is that if I use the default 'H&E DAB' vector set
> for Landini's demo image or any of my images, I get what appears to be
> much more accurate hematoxylin and DAB channel images.

Maybe it looks accurate, but the idea is that one first defines the colours
used to make sure that the unmixing result is what one intends. I have also
noted that DAB does vary across labs but also the haematoxylin is quite
variable (specially since there are several methods).

Make sure that you colour-correct all the images (by dividing the original
image by the brightfield and multiplying the result by 255; you can use the
Calculator_Plus for doing it in one step without having to resort to 32bit
conversion).

> The DAB and hematoxylin vector values are the same for both vector sets,
> the only difference are the 'zero' vs eosin third vector sets.

In the H DAB option the third component is calculated as the orthogonal to the
other 2 available (some shade of green). In the H, E, DAB one the 3rd is the
eosin colour, so the transformation is forced to separate into those 3
instead. (that means that the 2 colour spaces are not the same). I guess that
this difference (by luck?) compensates for any inaccuracies that there may be
in the vectors.

> if I can use the default H&E DAB set as a better rough+ready vector set
> (especially for images where I don't have individually stained slides to
> determine the ideal vectors.)

The problem is, I think, that one is never sure that the other 2 vectors get
separated correctly; they may  have some degree of mixing between them. If
staining is 'exclusive' then you would see the effect, but if you have
co-localisation of the colours it may be difficult to realise the size of the
error, despite that you have an almost empty "3rd image".

If single-stained images are not available, you could try to define the
vectors on areas which are uniquely labelled with each stain, but
unfortunately one cannot make it always sure that this is so.

I hope it helps.

Cheers,

Gabriel