Hello image analysis experts,
I manage a small confocal core facility and am trying to help someone with their image analysis. The desired workflow is to collect multi-channel z-stack images on our Nikon confocal (.nd2 files, 12bit), deconvolve data in Huygens Professional (here she is saving as 16 bit tiff files), and then colocalization of two channels (red and green) with the coloc2 plugin. However, we have run into some issues with the deconvolved files taking excessive amounts of time to analyze in FIJI.
If I test ImageJ’s preloaded sample images, the Coloc2 plugin works relatively quickly for both single planes and z-stacks. Similarly, if I load the original nikon z-stack files (which read into bioformats as 16 bit), the analysis takes a reasonable time for a ROI around a cell (~5 minutes). However - if I load the deconvolved images, and use the same ROI - the analysis takes over an hour per cell, even on well equipped analysis workstation computers. It seems to get stuck on step 2 of 11, running auto threshold regression. I have updated FIJI and ensured that the allocated memory is sufficiently high. With user permission, I have uploaded sample images to Google drive - please let me know if you have any trouble accessing them:
My understanding is that the colocalization of red and green signals is not expected to be great biologically in this example, but the desired analysis workflow remains.
Within the Coloc2 plugin, we are currently using the default settings as we are new to the plugin, but any advice on how to optimize the analysis would also be greatly appreciated.
Does anyone know why these deconvolved images should take so long for a single ROI analysis in Coloc2?
Thank you in advance for any and all advice!