CellProfiler for smFISH dot counting

cellprofiler

#1

Hi
I’d like to use CellProfiler for the quantification of single-molecule fluorescence in situ hybridization (smFISH) for mRNA quantification in tissues. Most existing tools use two-dimensional Gaussian fitting of candidate spots to obtain a sub-diffraction localization of single mRNAs (eg http://www.nature.com/nmeth/journal/v10/n4/full/nmeth.2406.html). So far I haven’t found any implementation of such algorithms for CellProfiler, does anyone have any inputs?

Thanks
Andreas


#2

Superresolution/single molecule algorithms are on our roadmap but to my knowledge they’re not ready yet. We’re going to be increasing our capacity for implementation of new algorithms soon though, so stay tuned!


#3

Any progress with the single molecule counting implementation?I couldn’t see them on your roadmap when I checked last time.

Thanks


#4

Any progress with the single molecule counting implementation?I couldn’t see them on your roadmap when I checked last time.

It was on the version of the roadmap we had early last year, it got taken out when we streamlined the roadmap in late August. Sorry!

The short version is no, that’s not something we’ve been working on lately because of our big push for 3D support, but it’s still under consideration. Once 3D is finished we’re planning to sit down and prioritize what modalities come next; I’ll keep this request in mind.

In the meantime, apparently some people are trying to adapt CP as it currently exists for superrresolution, so hopefully that workflow gives you some interesting ideas!


#5

Hi, just checking in if the implementation of single molecule counting / local maxima identification is on the roadmap for upcoming versions.

Thanks


#6

So far our new roadmap is more focused on changes to the software rather than particular biological implementations, simply as that’s necessary to keep the software ongoing and upgradable in the future.

All that being said, all of the steps in the paper you linked are either already implemented (or should be implementable) in CP 3.0, so it’s definitely possible to create a module and/or pipeline that would do this. I’d start by trying to make a pipeline by looking at the steps in the workflow and matching them to current CP functions; if you’ve got a few images analyzed with an existing tool you can use for “ground truth” counts, you can then compare them with your pipeline’s output. If you get stuck at any point, you can post a pipeline and images and we can try to help you troubleshoot!