I want to quantify fluorescence and co-localization in microscopy images from some IHC experiments using ImageJ, but am unsure of how to do so. If anyone has a detailed protocol or could direct me to one, I would appreciate it
Dear Jordan, at image j website you have several pluggins to do that. To calculate the intensity you can obtain the medium value of your signal in your picture just using the measurement command of the program, setting flirts what you want to measure, the relative intensity would be fine. For coloc you can use pluggins such jacop or similar. Good luck
You might also wanna try CellProfiler software. Not as customizable as ImageJ, yet still has great potential.
This is what I do for DAB quantification on slides with hematoxylin counterstain:
Download the free "Fiji" version of ImageJ from http://fiji.sc. All subsequent steps are performed in Fiji.
Open a DAB image.
Run Image > Color > Colour Deconvolution.
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).
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.
Run Analyze > Set Measurements and select "Mean gray value" and "Display label".
Select the Colour_2 image window.
Run Analyze > Measure (or press Ctrl-m); a "Results" window will pop up with the quantification in units of intensity.
You need to convert the intensity numbers in the Results window to Optical 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.
Not sure if it's the best way, but this is how I do it:
In imageJ, circle all the cells so that each one has an ROI. You can save this ROI and use it for the other colours you have in a different image. Click measure after circling each one (ctrl+T i believe). Average the three cells with the lowest intensities, this is your background. then calculate the threshold from that as twice the background intensity plus 6 times the SEM of average background.
I hope that helps
Fiji is the same as imageJ but bundling a lot of plugins which facilitate scientific image analysis.
I used many image analysis softwares and compared between them to ensure my results. So you can download my publications and use them as a guide and a reference for your work.
https://www.researchgate.net/profile/Hesham_Mustafa.
Check this book: https://www.researchgate.net/publication/260261544_Analyzing_fluorescence_microscopy_images_with_ImageJ
Hi everybody, I would like to to quantify cell density after H&E staining with this method.
Does anyone help me? I read the method for DAB...what's happen with H&E?
Thanks a lot,
Jessica
Dear @Hesham Mustafa. There is any other way to present the results? Could I have the quantification of positive cells / area? Other question, I have some slides with less tissue than others, could you explain me how can I use Integrated Density instead Mean. Thanks
Dear Nancy Marin.
There is any other way to present the results?
Yes, there is alot of ways to present the results depending on your goal.
Could I have the quantification of positive cells / area?
Yes, but please follow a reference (search online you will find alot of references according to your goal) (every step should be supported by a reference and a trial using 2-3 different softwares will help and a simple pilot study for the counting this will help).
quantification of positive cells / area could be done as a percentage especially for the biological tissues.
Other question, I have some slides with less tissue than others, could you explain me how can I use Integrated Density instead Mean.
Sure, in the biological sample you may face this problem and the solve to use (AOI) area of interest in every software you can delineate your desired area of interest (and this is the key).
Again you have to support your results with references.
Hello @Hesham : thank you vey much for your information. Could you explain a little bit more about what to do when the image has areas without tissue? Tank you very much. you are already my hero.
Dear Sonia H. Navia replying to your email in measuring the area of interest (AOI) to solve the problem of missing tissues, please read carefully the following link and you will find the answer of your inquiry
https://imagej.nih.gov/ij/docs/guide/146-10.html
One thing to add is that "Mean gray value" that the program gives seem to be given as 255-x (numbers close to 255 = images closer to white). So to analyse the data one should first subtract the values given by the program from 255. Then the Mean intensity can be quantified as (255-"Mean gray value") and OD as log(255/(255-Mean intensity)). The numbers for OD will be the same. What also can be done: the same procedure can be performed for the stain-negative areas to quantify the background that can be subtracted.
Hi,
I recommend to read the following link:
https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html
Hesham N. Mustafa Thanks for your advice re OD, this is really helpful. Do you happen to have a reference for its use in IHC quantification or at least direct me to one? Thanks again
dear Jordan, here's what you need
https://imagej.nih.gov/ij/plugins/ihc-toolbox/
good luck
Hi, how do you measure percentage of positive cells on a DAB image? Which formulas/macros do you recommend on Image J?
Thanks!
Hesham N. Mustafa Suppose the OD comes out to be 0.1, 0.5 or any other value like 1 or 2 ( i am just assuming these values) then what does that mean? Also how do we calculate the H-score?
We published some years ago something reated to this. Maybe that helps.
Here is available a protocol for Semi-quantitative analysis of IHC images using ImageJ Fiji:
Bio Protoc. 2019 December 20; 9(24): . doi:10.21769/BioProtoc.3465.
Hi,
I recommend to see the following link:
https://www.youtube.com/watch?v=gxnWYhZ0z4k
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