Extended Particle Analyzer- Problem counting particles

Hey all,
after having used ImageJ Particle Analyzer to count Collembola in scanned petri dishes, i found the Extended Particle Analyzer. With this plugin i could define further parameters to “clean” up my counting results. However, when i execute the plugin with default settings it only returns a blank white “drawing”-window without any or with only one result. Ive tried binary images with white objetcs/black background as instructed but nothing…

I would really appreciate it if somebody would take the time to help me out.
Thank you!
Alex

PS: This is a scan i am working with

Hi @Deibel,

could you probably do your processing again as you did before and record it with the macro recorder (→Plugins→Macros…→Record…) and then paste the code here, just to get an idea, what exactly you did especially before you ran the Extended Particle Analyzer. This might help in finding a solution.

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Hi, thank you for your reply!

Macro Recorder:
selectWindow(“tif044.tif”);
run(“Subtract Background…”, “rolling=150 light”);
run(“Enhance Contrast…”, “saturated=0.3”);
setAutoThreshold(“Default”);
//run(“Threshold…”);
//setThreshold(0, 225);
setOption(“BlackBackground”, false);
run(“Convert to Mask”);
run(“Close”);
run(“Invert LUT”);
run(“Extended Particle Analyzer”, “pixel show=Outlines redirect=None keep=None display summarize exclude include”);
selectWindow(“tif044.tif”);

selectWindow(“Drawing of Mask of tif044-1.tif”);

Hi @Deibel,

I think the problem in your case is that your your background is set incorrectly. The macro contains the commans setOption(“BlackBackground”, false);. That implies that Fiji takes white as background and analyzes black areas. Please, set the background under ►Process►Binary►Binary options… and activate the black backgroundcheckbox.

Hope that solves the issue

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It worked! Fantastic! Thank you so much!

Cheers!

Unfortunately it only worked for a couple of hours. Now I am back to an empty drawing and no results without knowingly having changed anything. Does that happen?

Cheers!

When it happens, check first if the black background option is still active. If not, that would be the problem. Then, another tool might change this setting. Is unlikely, but can happen. Can you potentially post (or send me) an example image (as png or tiff), in which it does not work. You can in parallel test on those images the normal Analyze Particles… function to see, if the same error occurs there. If yes, it is a problem with the image or a setting in ImageJ. If not, it might be related to the Extended Particle Analyzer.

The black background was active. I unchecked it and checked it again to see if it would help, but it didnt. Doing the parallel test the Particle Analyzer always returns results while the extended PA does not.
I have tried it on different Images without success. My Scans are large (ca. 400 mb) How can I send one to you?

Cheers!

Just cut out a square area around 500-1000 to test it on a smaller version

Just tried that one myself. Didnt work.

Thank you for your help!

Actually, for me it works on that image part. here are the steps I used orienting on your macro from above.

run("8-bit");
run("Subtract Background...", "rolling=150 light");
run("Enhance Contrast...", "saturated=0.3 normalize");
setAutoThreshold("Default");
setOption("BlackBackground", true);
run("Convert to Mask");
run("Extended Particle Analyzer", "pixel show=Outlines redirect=None keep=None display summarize exclude include");

tried your macro on several of my images. Unfortunately still nothing!.

And does the binary image show your insects extracted in white when you use the Default threshold?
Are you sure, that none of the parameter fields has any limitation which excludes the objects during the analysis. I would run it with the checkbox Reset after analysis activated once and then try it again, so that all the parameters are reset to default. Just to exclude that one of the parameters accidentally set is the culprit.
Since I cannot reproduce the problem, it is not an EPA intrinsic problem (at least does not look like that)

resetting the parameters worked! Now looking for the culprit!
Really appreciate your help!
Cheers!

Then you surely had one of the multiple parameters changed and that excluded that objects. That should be solved now :smile: