Measuring small nuclear foci

Hello! I’ve been using the count speckles pipeline to count my nuclear foci. I think I’ve gotten it to a point that it is useful but I’m still having problems with how it detects the actual foci. I know it has to do with thresholding and size of the foci but I’ve tried to figure it out by reading all the foci entries and looking at some of your tutorials but I’m still lost.
Here is my pipeline. I haven’t changed much from the original one (although I’ve tested a bunch of different parameters but I got lost, hence why I’m here)
FociTest1.cpproj (598.5 KB)
Here are two of my images


This image should have very few speckles, but I get a massive number. Also, I do not understand what the image below is showing me. I the window named outlined all the speckles and then the one labeled RAD51Foci just the ones that pass the test?


Any help is greatly appreciated! As I said I read most of the other posts that had to do with Foci, but clearly I’m missing something!

thanks

Hello! I believe your parameters just need adjustment.

Either use a EnhanceOrSuppressFeatures feature size of 4 and raise your IdentifyPrimaryObjects lower diameter limit to 2 px OR use a EnhanceOrSuppressFeatures feature size of 3 and keep the lower diameter limit at 1. The first option will also identify smaller foci, the second will only recognise the most prominent (I got 24 foci in your test image)

Concerning the image you are asking about: The IdentifyPrimaryObjects module will present you with limited results in this window. The “Input Image” is simply the image you are using. The “Outlines” will overlay object outlines on your Input image - green outlines are objects that fall within your size restraints, purple are objects that are rejected because they are too large/too small (yellow objects are discarded for touching the border). The third window shows a black background (not your image) with differently colored objects that HAVE been accepted, as you have guessed already.

Good luck!

Hello!
Thank you so much for your suggestions! I’ll try them today.
In terms of the IdentifyPrimaryObjects module, I take it that the third window doesn’t show all the foci? Because I never see the number that the program tells me have been accepted in that window, hence my confusion. Is there a way to have an image with the foci highlighted as part of the pipeline? I know my lab will ask me for such image!

Thanks again!

Monica

When I execute IdentifyPrimaryObjects, I see this window:

1 - Your input image
2 - ALL detected foci (including those too small or too large), those within the accepted range are outlined in green.
3 - A black background with your ACCEPTED objects highlighted in different colors.
4 - a table with module results and parameters. You see the “# of accepted objects” (66 in this case, using your original settings), info about size of your objects, as well as some module parameters (like Threshold, smoothing, etc).

Saving the image does not save this table - and saving the table does not save the image. Using “Alt+PrintScreen” you can copy the entire window and paste it into an image editor of your choice.

I hope this answers your question!

p.s. Don’t forget - there is a “test mode” that lets you move through your pipeline by clicking “Step”. Changing one of the parameters of a module will reset your current position in the pipeline to the module you have altered.

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Thanks for all your help. I was able to find a way to save the image with the chose objects, which is what I wanted. I ran it on the test mode and it seems to work. Now I’m working on batch image processing which I think is going well.

Thanks!

Monica