(IMPORTANT) **BIOP BioImage analysts DO NOT recommand to measure intensities of DAB.** = BIOP recommandation= == Color Deconvolution + Automatic Thresholding== We recommand to use the [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | color deconvolution]] plugin for Imagej to separate colors into channels, Then use a [[ https://imagej.net/Auto_Threshold | automatic thresholding]] to generate binary images, So we can measure the **Area** of the **Staining of interest** AND of the **Tissue**. {F5833801, size = full} == Why measuring Tissue Area?== {F5833808, size = full} =Why you should not measure DAB intensities ?= (IMPORTANT) **Because DAB intensities are not linear ! ** == From the authors of the [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | 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."// == From BIOP test == == Fluorescence intensities are linear on a widfefield microscope Beads with known intensities (100% , 30%, 10%, 3%, 1%, 0.3%, 0) {F5834173, size=full} ...were acquired on a widefield microsocpe (16-bit camera) {F5834182, size=full} ...measured with a ImageJ script {F5834196, size = full} == What About DAB ? == We mesure intensities of individual cell on the very same slice (//not on consecutive slice**s**//). {F5833997, size = full} Extract Fluorecsence channel and use [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | color deconvolution]] plugin to get the DAB component {F5834003, size = full} Detect the cell using the DAPI ... {F5834016, size = full} ... and **measure intensities** on both Fluorecsence or DAB component {F5834024, size = full} ... and if you compare for each cell its value from each image {F5834046, size = full} You realize that DAB are not linear.

(IMPORTANT) **BIOP BioImage analysts DO NOT recommand to measure intensities of DAB.** = BIOP recommandation= == Color Deconvolution + Automatic Thresholding== We recommand to use the [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | color deconvolution]] plugin for Imagej to separate colors into channels, Then use a [[ https://imagej.net/Auto_Threshold | automatic thresholding]] to generate binary images, So we can measure the **Area** of the **Staining of interest** AND of the **Tissue**. {F5833801, size = full} == Why measuring Tissue Area?== {F5833808, size = full} (IMPORTANT) You can use this [[ https://c4science.ch/w/bioimaging_and_optics_platform_biop/image-processing/imagej_tools/ijab-biop_ihc_ratioquanti/ | tool ]] to do Areas Analysis. =Why you should not measure DAB intensities ?= (IMPORTANT) **Because DAB intensities are not linear ! ** == From the authors of the [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | 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 [[ https://list.nih.gov/cgi-bin/wa.exe?A2=ind0902&L=IMAGEJ&P=R18412 | 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) {F5834173, size=full} ...were acquired on a widefield microsocpe (16-bit camera) {F5834182, size=full} ...measured with a ImageJ script {F5834196, size = full} 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". {F5833997, size = full} Extract Fluorecsence channel and use [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | color deconvolution]] plugin to get the DAB component {F5834003, size = full} Detect the cell using the DAPI ... {F5834016, size = full} ... and **measure intensities** on both Fluorescence image or DAB image {F5834024, size = full} ... and if you compare for each cell its value from each image {F5834046, size = full} You realize that DAB are not linear.

(IMPORTANT) **BIOP BioImage analysts DO NOT recommand to measure intensities of DAB.** = BIOP recommandation= == Color Deconvolution + Automatic Thresholding== We recommand to use the [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | color deconvolution]] plugin for Imagej to separate colors into channels, Then use a [[ https://imagej.net/Auto_Threshold | automatic thresholding]] to generate binary images, So we can measure the **Area** of the **Staining of interest** AND of the **Tissue**. {F5833801, size = full} == Why measuring Tissue Area?== {F5833808, size = full} (IMPORTANT) You can use this [[ https://c4science.ch/w/bioimaging_and_optics_platform_biop/image-processing/imagej_tools/ijab-biop_ihc_ratioquanti/ | tool ]] to do Areas Analysis. =Why you should not measure DAB intensities ?= (IMPORTANT) **Because DAB intensities are not linear ! ** == From the authors of the [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | 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 [[ https://list.nih.gov/cgi-bin/wa.exe?A2=ind0902&L=IMAGEJ&P=R18412 | post ]] within imagej archive. == From BIOP test == == Fluorescence intensities are linear on a widfefield microscope BWe mesured intensities of beads with known intensities (100% , 30%, 10%, 3%, 1%, 0.3%, 0) {F5834173, size=full} ...were acquired on a widefield microsocpe (16-bit camera) {F5834182, size=full} ...measured with a ImageJ script {F5834196, size = full} That appears to be //quite// linear. == What About DAB ? == We mesured intensities of individual cell on the very **same slice** (//not on consecutive slice**s**//)es//) stained by "fluorescenct probe" then "DAB". {F5833997, size = full} Extract Fluorecsence channel and use [[ http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html | color deconvolution]] plugin to get the DAB component {F5834003, size = full} Detect the cell using the DAPI ... {F5834016, size = full} ... and **measure intensities** on both Fluorecsencscence image or DAB componentimage {F5834024, size = full} ... and if you compare for each cell its value from each image {F5834046, size = full} You realize that DAB are not linear.