Problem with IdentifyPrimaryObjects for nuclei. Only see streaks of lines... Does not circle nuclei

I’m using a pipeline on my Windows 64-bit machine. The first analysis module of the pipeline is IdentifyPrimaryObjects. I’m trying to identify nuclei with this, but every time I run the module, it comes out with a streak of lines over my cells instead of circles around the nuclei.

Someone else ran the exact same pipeline on her computer (Mac 64bit) with the same images and it worked perfectly.

I’m imaging NIH 3T3 fibroblast cells. The goal of the entire pipeline is to outline cells and measure antibody fluorescence intensity. But I can’t get past the first step!

Here’s the pipeline:antibodyfluorescence.cpproj (96.2 KB)

That can happen sometimes with the declumping method set to Shape- try setting it to something else (or turning declumping off if all your images are this sparse) and that shouldn’t happen anymore.

And if that doesn’t work, include the images that go with the pipeline! (the fact that this is specific to a particular computer is pretty disturbing/suspicious!)

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Thanks for the quick replies!

We realized that there was a small difference in the images analyzed on each computer. Sorry for the confusion. The original images are z-stacks. My friend made the images into z projections of the maximum intensity in order to analyze them, whereas I made z projections as the sum of all the slices. This made her image 16-bit but mine 32-bit. Turns out the module doesn’t work with 32-bit.

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Wow, great sleuthing!