Does anyone have recommendations for a blue fluorescent protein that’ll work well for SIM (specifically with the OMX)?
Apparently a while ago a GE rep suggested TagBFP. A user spent months going through the whole process of tagging a lamin with it before chatting with me and seeing how poorly matched the OMX’s DAPI emission filters are to TagBFP. Looking at the FPbase efficiency report fills me with despair.
Her cells have been selected by FACS, so it’s not an issue of continuously selecting cells with weak TagBFP expression.
TagBFP(/2) definitely sucks for SIM, as you’ve observed… but I don’t know of a better option.
Just in case it hasn’t already been considered, and if you’re not doing live imaging, perhaps you can use the TagBFP as an epitope for antibody-based amplification with some better organic dye (possibly even in some other wavelength). Given it’s lineage, a TagRFP antibody might work.
but if you strictly require a fluorescent protein and it has to be in the 405ex channel… i don’t know what else to suggest…
I should have mentioned this in the initial post, but her panel currently consists of TagBFP, EGFP, TagRFP, and miRFP670. The other channels look great.
In her experiments, “antibody detection is not an option!”
Life is hard.
I’ll keep your amplification suggestion in mind for other users with more flexibility.
Oof, that is tough. Could you do a Halo- or Snap-tag? I’m not sure what the best fluors are for that in the BFP range but they are likely to be better than TagBFP
Yeah I think we need more details on the experimental constraints. Are antibodies not an option because no good primary exists to the protein of interest? Because of cell permeability in living cells? Labeling density/efficiency requirements? There is likely an alternative strategy available… but it depends on the constraints.
I recommend Halo- or SNAP-Tag in combination with JaneliaFluor ligands. There are a wide variety of fluorophores available. It should not be a problem to find dyes that work well with your filter sets.
I would suggest using a UV-excitable GFP. Excitation max. is at 399 nm, but it is still efficiently excited at 405. Emission is identical to GFP.
Co-imaging with ordinary GFP can be achieved by switching the excitation source. The only downside is potential cross-talk, although excitation of UV-excitable GFPs at 488 is minimal.
The best variant that is published is mT-sapphire. We have recently made an upgrade of that; LSS-SGFP2 (large Stokes-shift SGFP2). It is 2-fold brighter (unpublished) and available from addgene.org: https://www.addgene.org/browse/article/22612/
Is it possible to try upconversion nanoparticles? These have multiple advantages but would depend on the optics of the OMX system. Or quantum dots … With nanoparticles or quantum dots you have to have an affinity tag so it can localize properly…so that’s harder if no antibodies…TagBFP and other blues are typically dim and bleach easily, the UV excitable GFP is a good idea from Joachim. If you can do some spectral separation then CFP or YFP could even work, or, get some new glass filters with special cut-offs and excite off peak (short on the CFP, long on the YFP) and image the short part of the CFP emission or long part of the YFP emission to limit bleeding into the GFP. Chroma and perhaps the other companies will work with users to do some custom filter building. Good luck!