Setting different intensity thresholds for different spot channels?

Hi all,

I am trying to use starfish to automate analysis of RNAscope images (12-plex HiPlex version) that involves images from 3 imaging rounds with each round including a DAPI, FITC, Cy3, Cy5, and Cy7 channel z-stack.

My current workflow allows me to z-project, register, and filter out background on all channels at once, but I am having a hard time reliably detecting spots across all channels because the intensity of the Cy7 spots is so much lower than the other channels. A low intensity threshold that cuts out background in FITC also cuts out the real signal in the Cy7 images. I was wondering if there is a way to set different intensity threshold for each channel? I am currently detecting spots with the FindSpots.BlobDetector function and I don’t see anything in the documentation about setting different thresholds for different channels. Thanks!

Hi thanks for your great question.

You’re right there is no way to set different intensity thresholds in the FindSpotsAlgorithms. Instead, the image processing pipeline uses intensity normalization methods to normalize images from different channels. I think you may find the various clipping filters that also scale the intensities helpful.