7/14/26 edit: Putting this edit up top so it’s noticeable. But Dr. Nick Coleman (PI that developed fuGFP!) send me a very nice email informing me of an oversight on our end when we did the experiment.
“…I was surprised that the fuGFP performed so poorly, since in our hands it seemed very bright (although to be fair we never compared it quantitatively side-by-side with other GFPs). I was curious whether you used the same excitation source for all the fluoroproteins in this experiment or whether you tuned this depending on their preferred excitation wavelengths.The fuGFP much prefers a long-wave UV light source rather than a blue light source – could this explain its poor performance in this assay?…”
He was completely correct. In retrospect, this detail is actually pretty clearly noted in FPbase (my go-to for FP information). Indeed, I checked our notes and we excited it with blue / 488nm laser light as we did with all the other green FPs, so that was an oversight on our end. Thus, it’s quite likely it would have been much brighter had we excited it as intended. Aside from an enjoyable short correspondence with someone who I’ve drawn some inspiration from (I mean, the justifiable irreverence and cleverness of fuGFP!), and some better refining my understanding / perspectives of fluorescent proteins, it was also a good reminder that perhaps this would be a fun target to try some directed evolution with HuMutaT7.
Start of original post:
At one point, I was a doe-eyed postdoc reading about new fluorescent proteins (FPs) with improved brightness and thinking it was potentially important to incorporate new FPs into my constructs for cell culture work. I have since come to realize that the intrinsic gains to fluorescence published in those papers do not necessarily translate to brightness in my experiments. The reason are probably multifactorial, including:
1. Commonly accessible equipment is generally optimized for EGFP (or similar FPs), so newer FPs with slightly different excitation and emission spectra may not be captured well with existing microscopes or flow cytometers.
2. The FP brightnesses are typically assess in-vitro, and there may be other factors in eukaryotic cell cytoplasms that may affect the FP brightness (eg. FP half-life).
Well, I’ve still ordered a handful of different green fluorescent proteins anyway, and figured it was worth doing a side-by-side comparison in the transgenic system we use in the lab. This was all done with the HEK293T G542Ac3 (LLP-Tet-Bxb1attP_Int-BFP_IRES-iCasp9-BlastR) cells, which were stably recombined with a single copy of each fluorescent protein at a common genomic locus. The construct organization was: Bxb1attB_[Green FP]_IRES-mCherry-2A-PuroR. Olivia did these recombinations, selected the cells, and ran the cells on the ThermoFisher Attune Flow cytometer, with Sarah’s help. This is what the results look like:
Some interpretations:
1. Rather minimal (~3-fold) difference between mGreenLantern and UnaG. I suppose if we were ever in a situation where we needed every unit of green brightness possible, that we would go with mGreenLantern. That said, UnaG has some benefits; namely, it’s 58% the size of EGFP/mGreenLantern/mNeonGreen, and it lacks the VERY ANNOYING identical sequences at the N- and C-termini (MVSKG … DELYK) which makes molecular cloning a potential pain.
2. Conceptually, I like the idea of fuGFP, but that 20-fold diminished green fluorescence compared to EGFP is potentially problematic. Who knows, maybe I’ll turn this into a target of directed evolution at some point…? **See my 7/14/26 note about the fact that it’s surprinsingly low fluorescent intensity was likely from exciting it at the wrong wavelength.
5/22/23 edit: We recently tested StayGold, and the results were rather underwhelming. In our n=1 experiment, it yielded a 6% increase over mGreenLantern in green MFI within stably expressing landing pad cells. If it were on the EGFP-normalized scale of the chart / experiment above, it would be a value of about 2.15. So ya, not that it’s not potentially better than mGreenLantern; it’s more that, not sure if it’s worth the effort for most applications.