Thank you both for your replies!
What I’m getting from these two posts is that the Xenium can plausibly localize the center of an isolated mRNA strand to <30 nm, due to the large number of photons generated by an RCA amplicon. Even if the diffraction spots are very wide due to the RNA being outside the optimal focal plane, the large photon count should rescue it?
However, if two strands from the same gene are near each other, the results might be inaccurate? Since they are only taking a single image of each cycle (I assume?), the Xenium likely cannot differentiate two overlapping PSFs from two strands? Would the amplicons interfere with each other if they were too close together? In a situation like this from @Daniel_Furth 's fantastic example image, would it be able to get a trustworthy count and localization?
Thank you again!