Re: lamp temperature

Posted by Gabriel Landini on Jun 03, 2011; 8:00am
URL: http://imagej.273.s1.nabble.com/lamp-temperature-tp3684358p3684359.html

On Thursday 02 Jun 2011 19:04:27 you wrote:

> Hello- I am looking for advice on whether I can correct microscope images
> for variations in lamp filament temperature when analyzing slides for
> stain intensity. In a first batch of slides, the background was white, the
> (unstained) tissue was tan and the stain blue.  Using the Landini
> thresholding plugin to select for blue hues followed by measuring the mean
> saturation worked well.  However, in a second batch of slides - which were
> intended to be combined with the first in the analysis - the slide
> background and the unstained tissue are a yellowish color and the stain
> hues are in the blue-magenta region.  That creates a problem in separating
> the tissue from the background, and also the mean saturation of the hues
> attributable to the stain are different from the first batch.  Is there a
> good way to correct for lighting variation in these two image sets, that
> would allow an accurate and consistent stain intensity to be quantified?

It is possible to compensate for that and uneven illumination at the same
time. This is the traditional way of compensating illumination via
transmittance:
http://imagejdocu.tudor.lu/doku.php?id=howto:working:how_to_correct_background_illumination_in_brightfield_microscopy

The other problems with stains are:
The colour threshold plugin would work only if the stains do not colocalise.
For instance it is virtually impossible to be able to threshold H&E this way
because many pixels will record a combination of both dyes.

You might want to try stain separation using colour deconvolution, but be
aware that if the stains are not stoichiometric (most are not) then the
intensity of the stain will not be a quantitative measurement.

Cheers

Gabriel