I am trying to analyze some ISH images but I am not sure how to get the total area with a positive signal for my target-specific probe. I am looking for CXCL13 in lung tissue. Any help would be welcome!
Would you be able to post an example image? That would help us help you. And just indicate what you consider positive signal…
Here is one of those images. I need to calculate the area occupied by the dark dots compared to the total area. Thank you for your help!
So… I just opened your image in ImageJ… and I’m worried about a few things:
Your image is a JPEG. Ideally - you should not be saving images as JPEGs - due to lossy compression - you can read more about that here. You should never use JPEG - especially for quantification analysis. It’s better to use TIFF or the original file format of your imaging system.
If you zoom in on your image - you will see that the resolution of your dark spots is not high enough to measure reliably. Pixel-sized dark spots are abundant… so there is no way to say that they are real or noise or what? At least - I’d not consider this resolution reliable (perhaps others on the Forum here would disagree?) Have a look at the image below that I took of yours zoomed in. As it is almost impossible within this image to delineate your individual cells - I would doubt you could reliably measure your CXCL13 staining within.
All-in-all … would it be possible to re-acquire these images at a higher resolution and saving as TIFFs??
I know this is perhaps not the answer you’d wanted… sorry for that. But it’s the best way to get the best data you can.
Thanks for your comments. I have the images in .tiff and I posted one for you here (20X magnification). I have the images with a high quality because they were acquired using the nanozoomer but I need to figure out how to do the analysis using imageJ.
I would like to ask you what is your suggestion for doing this analysis? I was trying to use imageJ with best quality but it seems like the numbers that I got correspond to the intensity of pixels but no the area corresponding to the specific staining.
I really appreciate your help!