Quantitative comparison of fluorescent proteins using protein nanocages in live cells

To standardize comparison of fluorescent protein performance on a
molecule-by-molecule basis in a physiological intracellular environment,
we constructed fluorescent protein tagged I3-01 peptides that
self-assemble into stable sixty subunit dodecahedrons inside live
mammalian cells. We were especially interested in determining which of the
recently published monomeric StayGold variants is best for live microscopy
in mammalian cells. Combining nanocage brightness and photobleaching
measurements into a single metric, mStayGold stood out as far superior to
all other green and red fluorescent proteins we tested with a functional
lifetime that is at least 8-10-fold longer compared with EGFP or mEmerald.
Analysis of intracellular nanocage diffusion further confirmed the
monomeric nature of mStayGold and we demonstrate that mStayGold-tagged
nanocages can serve as highly photostable nanoparticles to analyze
intracellular biophysical properties. Analysis of frequently used red
fluorescent proteins was less encouraging and recent mScarlet or mRuby
variants did not perform substantially better than mCherry on a typical
spinning disk confocal microscope system, highlighting the importance of a
standardized method to benchmark fluorescent proteins to make optimal
choices for specific experimental setups.