Electronic Automated Measurement User System (EAMUS™)

Stereology

The stereology measurement program provides you with general quantitative data of an immunohistochemical image. It is suitable
for low magnification images, and can distinguish (counterstained) nuclei from other biological compartments. It can be applied
to conventional immunohistochemistry (DAB, AP), and fluorescent images (DAPI, FITC, Texas red).

1. In non – fluorescent (DAB, AP) images the measurement will provide you with the following data:

Measurement data

Vv nuclei neg: Volume fraction of epithelial cells without detectable staining (according to formula Vv=100*k*S{(pixepi)/(pixtot)}
Vv positive stain: Volume fraction (Vv) in percent of positive stained pixels (independent from structure or segmentation)
% pos nuclei: percent of positive stained Vv nuclei Vvnucpos/Vvnucltot;
% weak stain: Percent of weakly stained volume fraction (Vv)
% mod. stain: Percent of moderately stained volume fraction (Vv)
% inten stain: Percent of intensively stained volume fraction (Vv).

Remarks: The intensity is calculated according to the gray value total of nuclear area (Integrated optical density, IOD).
Weakly stained: <34% of maximum IOD;
Moderately stained: >33% - <67% of maximum IOD
Intensively stained: >66% of maximum IOD (usually 256 gray values).

2. In fluorescent (TEXAS – RED, FITC, DAPI) - stained images the measurement will provide you with the following data:

Measurement data

red fluorescent

%o Vv negative: Volume fraction of non – stained area in the red color space (TEXAS RED), (according to formula
Vv=100*k*S{(pixepi)/(pixtot)}
%o Vv pos. weak: Volume fraction of the weakly stained area (weakly/total stained) area
%o Vv pos. mod.: Volume fraction of the moderately stained area (moderately/total stained) area
%o Vv pos. strong: Volume fraction of the intensively stained area (intensive/total stained) area

green fluorescent

%o Vv negative: Volume fraction of non – stained area in the green color space (FITC), (according to formula
Vv=100*k*S{(pixepi)/(pixtot)}
%o Vv pos. weak: Volume fraction of the weakly stained area (weakly/total stained) area
%o Vv pos. mod.: Volume fraction of the moderately stained area (moderately/total stained) area
%o Vv pos. strong: Volume fraction of the intensively stained area (intensive/total stained) area

blue fluorescent

%o Vv negative: Volume fraction of non – stained area in the blue color space (DAPI), (according to formula
Vv=100*k*S{(pixepi)/(pixtot)}
%o Vv pos. weak: Volume fraction of the weakly stained area (weakly/total stained) area
%o Vv pos. mod.: Volume fraction of the moderately stained area (moderately/total stained) area
%o Vv pos. strong: Volume fraction of the intensively stained area (intensive/total stained) area

red + green fluorescent

%o Vv negative: Volume fraction of non – stained area in the (red and green) color space (TEXAS RED and FITC),
(according to formula Vv=100*k*S{(pixepi)/(pixtot)}
%o Vv pos. weak: Volume fraction of the weakly stained area (weakly/total stained) area
%o Vv pos. mod.: Volume fraction of the moderately stained area (moderately/total stained) area
%o Vv pos. strong: Volume fraction of the intensively stained area (intensive/total stained) area

red + blue fluorescent

%o Vv negative: Volume fraction of non – stained area in the red and blue color space (TEXAS RED and DAPI),
(according to formula Vv=100*k*S{(pixepi)/(pixtot)}
%o Vv pos. weak: Volume fraction of the weakly stained area (weakly/total stained) area
%o Vv pos. mod.: Volume fraction of the moderately stained area (moderately/total stained) area
%o Vv pos. strong: Volume fraction of the intensively stained area (intensive/total stained) area

red + green + blue fluorescent

%o Vv negative: Volume fraction of non – stained area in the (red and green and blue) color space (TEXAS RED and
FITC and DAPI), (according to formula Vv=100*k*S{(pixepi)/(pixtot)}
%o Vv pos. weak: Volume fraction of the weakly stained area (weakly/total stained) area
%o Vv pos. mod.: Volume fraction of the moderately stained area (moderately/total stained) area
%o Vv pos. strong: Volume fraction of the intensively stained area (intensive/total stained) area