Hi, I'm looking for a protocol for analyzing my bright field images stained with DAB and hematoxylin counterstain. I want to take into consideration the background DAB stain and my true DAB signal from the cells I am examining. Do I take the ratio of these intensities? Do I literally "subtract" the background intensity from my cell intensity? What do I do with these numbers from ImageJ? Images are taken under controlled settings but there is high variability from one tissue to the next. Also, I understand DAB is not meant for quantification of intensity but this material already exists and I want to work with it. Please advise and thank you!
This is what I do for DAB quantification on slides with hematoxylin counterstain:
1. Download the free "Fiji" version of ImageJ from http://fiji.sc. All subsequent steps are performed in Fiji.
2. Open a DAB image.
3. Run Image > Color > Colour Deconvolution.
4. From the Vectors pull-down, choose "H DAB" as the stain (this assumes your images are correctly white-balanced, otherwise you have to define your own colors by choosing "From ROI" for the vector then drawing your own Regions Of Interest to define the stain colors).
5. Click OK in the Colour Deconvolution window, you will get three new images. The one with "Colour_2" in the title is the DAB image (Colour_1 is the hematoxylin image), you will quantify the Colour_2 image.
6. Run Analyze > Set Measurements and select "Mean gray value" and "Display label".
7. Select the Colour_2 image window.
8. Run Analyze > Measure (or press Ctrl-m); a "Results" window will pop up with the quantification in units of intensity.
9. You need to convert the intensity numbers in the Results window to Opitcal Density (OD) numbers with the following formula:
OD = log(max intensity/Mean intensity), where max intensity = 255 for 8-bit images.
This will quantify the average darkness of the image due to DAB signal. If you have areas of the image without tissue it will bias your results because it will bring the average OD down. If that is the case we need to discuss further. Also, you may need to use Integrated Density rather than Mean.
Good luck and let me know if you need anything else.
You could use your background staining as a scaling factor for the dynamic range of the DAB. Assuming that the nonspecific background staining is linear with the specific staining (up to the point that the tissue is completely saturated), you can use the reciprocal of the intensity of the background to get a value that you will multiply the values of the specific staining by after you subtract the background. This should scale all of the images to the same intensity range.
You can check my publication (Savaris, ImageJ) in pubmed. for start.
The other is to measure the area, as in my paper
Ron's answer is great, but unfortunately I do not think it would work for DAB. DAB does not behave in a linear fashion, and calibrating it with the background would not circumvent the problem, because the assumption proposed would only be true for low-intensity staining as far as I know.
You can check figure 9 from
Van der Loos CM. Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging. Journal of Histochemistry and Cytochemistry 2008;56(4):313-328. doi:10.1369/jhc.2007.950170.
I have not read Ricardo's paper, but if there is another way to perform these measurements reliably it would be great!
Best of luck!
Hey everyone, thank you so much for the responses. I found a method for background illumination correction (http://imagejdocu.tudor.lu/doku.php?id=howto:working:how_to_correct_background_illumination_in_brightfield_microscopy) and using this method still gives me about a 10 unit different in the mean intensity of my 20x image (compared on 2 different light settings). Can anyone explain why? I was expecting to get the exact same means.
This is what I do for DAB quantification on slides with hematoxylin counterstain:
1. Download the free "Fiji" version of ImageJ from http://fiji.sc. All subsequent steps are performed in Fiji.
2. Open a DAB image.
3. Run Image > Color > Colour Deconvolution.
4. From the Vectors pull-down, choose "H DAB" as the stain (this assumes your images are correctly white-balanced, otherwise you have to define your own colors by choosing "From ROI" for the vector then drawing your own Regions Of Interest to define the stain colors).
5. Click OK in the Colour Deconvolution window, you will get three new images. The one with "Colour_2" in the title is the DAB image (Colour_1 is the hematoxylin image), you will quantify the Colour_2 image.
6. Run Analyze > Set Measurements and select "Mean gray value" and "Display label".
7. Select the Colour_2 image window.
8. Run Analyze > Measure (or press Ctrl-m); a "Results" window will pop up with the quantification in units of intensity.
9. You need to convert the intensity numbers in the Results window to Opitcal Density (OD) numbers with the following formula:
OD = log(max intensity/Mean intensity), where max intensity = 255 for 8-bit images.
This will quantify the average darkness of the image due to DAB signal. If you have areas of the image without tissue it will bias your results because it will bring the average OD down. If that is the case we need to discuss further. Also, you may need to use Integrated Density rather than Mean.
Good luck and let me know if you need anything else.
I developed a simple method using ImageJ to quantifying the intensity of DAB.
Here is the paper:
https://www.ihcworld.com/_books/Nguyen_Reciprocal%20Intensity.pdf
Here is the "How To" guide:
https://www.ihcworld.com/_books/Nguyen_Protocol_Reciprocal%20Intensity%20in%20Fiji.pdf
Dear all,
Did someone find the method used by Dr. Mustafa published or written somewhere? I would like to take a look to a more formal SOP or perhaps to a paper including this method.
Thank you
Hello there
I published the protocol, and it is quite well explained. PMID: 24341124
Fuhrich DG, Lessey BA, Savaris RF.
Anal Quant Cytopathol Histpathol. 2013 Aug;35(4):210-6.
Thank you very much, Dr. Savaris.
Just a quick question: do you think this method is still valid for tissue only stained using DAB (not stanined with hematoxylin at all)?
Thank you
I also have one question, if I have two tissue samples H&DAB and I calculated OD as mr Hesham described, and I got tissue A OD=0.165, tissue B OD=0.191, what does it practicly mean? Does it mean that I have more cells that stained in tissue B?
Thank you
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