Optimal Laser and Detector Filter Combinations for Fluorescent Proteins

The people at the CWRU flow cytometry core recent did a clean reinstall of one of their instruments, which meant that we had to re-set up our acquisition template. I still ended up eyeballing what would be the best laser / filter sets based on the pages over at FPbase.org, but I had a little bit of free time today, so I decided to work on a project I had been meaning to do for a while.

In short, between the downloadable fluorescent spectra at FPbase, as well as known instrument lasers and detector bandpass filters, I figured I could just write a script that essentially takes in whatever fluorescent protein with spectra that you have downloaded, and essentially makes a table showing you which laser + detector filter combinations give you the highest amount of fluorescence.

Here’s the script on the lab GitHub page. I made it for the two flow cytometers I use at CWRU (since, well, that’s where I work), although it would presumably be pretty easy to change the script to make it applicable for whatever instruments are at your place of work. So here’s a screenshot of a compendium of the results for the two main instruments.

^Above: Here are the results with the ThermoFisher Attune
^Above: and now also the results with the more familiar BD LSR2

Nothing too surprising here, although it’s still nice / interesting to see the actual results. After incorporating brightness metrics, it looks like I should be slowly shifting over more to using mNeonGreen, mScarlet, and TDsmURFP(!), though the differences are usually 2-3 fold, which in most cases won’t make or break an experiment.