Center for Relativistic Laser Science

GRC 2016

STORM (stochastic optical reconstruction microscopy), which offers an order of magnitude improvement in resolution, is based on high-precision single-molecule localization of photoswitchable fluorophores. The performance of STORM depends on photophysical properties of photoswitching fluorophores. Conventional fluorescent proteins used in STORM suffer from fast photobleaching, which results in limited number of independent snapshots in live-cell super-resolution imaging. We aim to develop a new STORM fluorophore for virtually no photobleaching. UnaG, a fluorogen-binding protein derived from the Japanese freshwater eel, fluoresces upon binding of nonfluorescent bilirubin, an endogenous metabolite. We discovered photoswitching behavior of UnaG: UnaG fluorescence turns off upon 488-nm illumination; UnaG in the dark state comes back to the fluorescence state upon 405-nm illumination. The photoswitching of UnaG can be repeated about hundreds of switching cycle. Most notably, we found that UnaG fluorescence recovers after photobleaching when exogenous bilirubin is added into the solution. We have applied UnaG to STORM imaging of the subcellular ultrastructure.

References

[1] A. Kumagai, R. Ando, H. Miyatake, P. Greimel, T. Kobayashi, Y. Hirabayashi, T. Shimogori and A. Miyawaki, Cell, 153, 1602 (2013).