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 60-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 disc confocal microscope system, highlighting the importance of a standardized method to benchmark fluorescent proteins to make optimal choices for specific experimental setups.