Hi everyone! I have a question regarding the processing of fluoscence microscopy images. I am trying to identify a rare population (CD3+ CD20+ CD19-) in a tissue rich of lymphoid (T/B) aggregates, where colours overlap too much to distinguish this population by eye also with greater magnification. Is there a way to subtract (make it "black" or blind) the signal only where CD19 and CD20 colocalize in order to better contrast any CD3+/CD20+ (if present) cell?
Please help!
Your microscope software should have a way to turn off specific channels. However, you can use image J (a free software) to split channels (colours) and then merge the selected color images together to have your specific CD19 and CD20 or any other. For more details follow these links. Goodluck
https://imagej.net/Color_Image_Processing
https://imagej.net/Colocalization_Analysis
Indeed, I really don't want to turn off a specific channel. What I need to do is:
a) to find areas of the tissue where CD20 and CD19 colocalize
b) to "blind" or remove that areas
c) to leave the CD20 signal only where it not colocalize with CD19 and to check if there are areas where it colocalize with CD3
This is because I need to identify cells that are positive both for CD20 and CD3 but not CD19; unfortunately i am dealing with tissue where there are abundant lymphoid aggregates of T and B cells and therefore is quite impossibile to find this very small population (probably isolated cells) by eyes.
Based on the information, I understood too many cells and hence too much and overlapping signal to detect the individual cells properly.
For this, I would suggest using Image J, where you can reduce the signal intensity (by adjusting brightness and contrast) to clearly see the differences in selected areas. You can also do this for selected colors to have a more clear observation.
https://imagej.net/Image_Intensity_Processing
And also see the attached picture.
If you can threshold the channels, then ImageJ's Image Calculator (see link below) would be ideal for this. It sounds like you need a sequence like this:
1. For each channel, threshold the image and make a mask.
2. To get mask of CD20 without CD19: CD20 mask minus CD19 mask
3. CD20 colocalized with CD3, but not CD19: mask from step 2 AND CD3 mask.
4. To overlay the result on the original image: click on the mask from step 3, Edit > Selection > Create Selection, click on the original image, Edit > Selection > Restore Selection. To save a copy of this outline, you can do Image > Overlay > Flatten.
https://imagej.nih.gov/ij/docs/guide/146-29.html#toc-Subsection-29.13
Could you possibly provide an example image? Our software has a fantastic tool for color deconvolution, which can be part of a larger recipe, perfect for handling challenging segmentation problems such as this! We've had much success using it for flourescence and histology applications.
Dear Theresa thank you. I preliminarily tried your technique and seems to work, although I need to optimize better the staining for improving quality.
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