It is often best not to use a counterstain when you need to threshold a
specifically stained feature. You can have duplicate slides made, one
set with counterstain, one without. If this is no longer possible you
can try to eliminate the affect of the eosin by using a red filter and
photographing your slides in monochrome mode.
Ralph Common
On 1/16/2011 2:13 PM, nwoller wrote:
> Hi,
>
> I am a new ImageJ user. I have taken a series of images of a slide with
> tonsil tissue which has been stained with hematoxylin as well as liquid
> permanent red (Immunostain). I have been careful to capture all of the
> images in exactly the same way, same light intensity, same exposure time,
> same white balance etc. I would like to count the number of
> hematoxylin-stained nuclei in my images. I threshold the image, apply a
> watershed and use "analyze particles". Unfortunately, I don't seem to be
> able to apply the same thresholds to my images. A threshold that works
> nicely for one image is completely hopeless for another image. It is
> possible that the intensity of the hematoxylin stain varies quite alot in
> the same tissue. A colleague therefore suggested that I should threshold by
> finding the top of the "threshold curve" and using that value for
> thresholding my images. What do you think? I would really appreciate some
> help and advice.
>
> Nina
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