II-a-2 Measure DAB intensity

Updated 1,096 Days AgoPublic

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BIOP recommandation

Color Deconvolution + Automatic Thresholding

We recommand to use the color deconvolution plugin for Imagej to separate colors into channels,
Then use a automatic thresholding to generate binary images,
So we can measure the Area of the Staining of interest AND of the Tissue.

Why measuring Tissue Area?

Why you should not measure DAB intensities ?

From the authors of the color deconvolution plugin.

A very frequently asked question in the ImageJ mailing list relates to quantification of immunostain intensity (for example DAB intensity) to evaluate antigen expression.
However, one needs to consider 2 main issues that prevent doing this in a quantitative manner:

• Antigen-antibody reactions are not stoichiometric, so "darkness of stain" does not mean "amount of reaction products".

In fact most histological stains are not stoichiometric (one exception is Feulgen stain which is commonly used for DNA cytometry).

• In particular DAB does not follow Beer-Lambert law. See, for example, CM van der Loos paper:

"The brown DAB reaction product is not a true absorber of light, but a scatterer of light, and has a very broad, featureless spectrum. This means that DAB does not follow the Beer-Lambert law, which describes the linear relationship between the concentration of a compound and its absorbance, or optical density. As a consequence,darkly stained DAB has a different spectral shape than lightly stained DAB."

an also see this post within imagej archive.

From BIOP test

Fluorescence intensities are linear on a widfefield microscope

We mesured intensities of beads with known intensities (100% , 30%, 10%, 3%, 1%, 0.3%, 0)

...were acquired on a widefield microsocpe (16-bit camera)

...measured with a ImageJ script

That appears to be quite linear.

What About DAB ?

We mesured intensities of individual cell on the very same slice (not on consecutive slices) stained by "fluorescenct probe" then "DAB".

Extract Fluorecsence channel and use color deconvolution plugin to get the DAB component

Detect the cell using the DAPI ...

... and measure intensities on both Fluorescence image or DAB image

... and if you compare for each cell its value from each image

You realize that DAB are not linear.