Hundreds of thousands of non-dots detected #15
Description
Hi! I am having an issue where RS-FISH is detecting hundreds of thousands of dots in images where we would anticipate only a few thousand at most.
Some information about our images:
We have three sample groups, probing for 6 3-gene sets per sample group, with three replicates (for a total of 9 images per gene set / gene); each sample is also DAPI stained. The images were taken with a Zeiss LSM 880 with AiryScan processing. Each image is very large, usually 12-15 tiles with a 5-z stack for each tile, with the three color channels and one grayscale channel. The laser intensity and gain for the "cyan" channel on one gene set is not always the same for the "cyan" channel on a different gene set, and so on for the other channels except DAPI, which is always the same laser power and gain. After imaging, we perform AiryScan processing, tile stitching, and a maximal-intensity projection.
In FIJI, I split the channels for each image into its individual genes. I then use RS-FISH on a single gene, fine-tuning the parameters with a few images from that gene, and then use those parameters on the remaining images once they look acceptable. Given the variability in our expression levels and fluorophores, the parameters are different gene-by-gene.
However, we have come across a problem where the parameters I set work very well for most images for a gene, but then occasionally one or two images will have something like >200,000 dots detected. Visually we would expect only a few thousand at most, and indeed when we overlay the ROIs it appears that the majority of dots detected are what we would consider background fluorescence or appear to be nothing at all.
We are using RS-FISH with RANSAC; a standard support region radius (3), min. inlier ratio (0.1) and max error (1.5); and a spot intensity threshold of 10,000. Thus, the only parameters I change between genes are the sigma and threshold, which are typically between 0.80-1.00 and 0.0005-0.0015, respectively. The Gaussian refinement and background subtractions don't appear to fix this problem, and can cause other issues like runtime errors, so we don't use them.
We are trying to figure out if this is a matter of RS-FISH, or if there is something with our imaging that may need to be addressed. I am happy to provide more information or images.