Calculating 3D centroids in tiff image stack

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Hello!
I am trying to calculate the centroids of multiple spheres that change position as you scroll through the image stack. The goal is to get x,y, and z coordinates from these centroids. I can find and track the centroids when I read the file as a 2D image, but I have been unsucessful when trying to calculate the centroids when I read the full file in its entirety (512x512x96). Any advice would be greatly appreciated!

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