Higher resolution in localization microscopy by slower switching of a photochromic protein.
Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.
Mizuno
Hideaki
H
Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan. hideaki.mizuno@chem.kuleuven.be
Dedecker
Peter
P
Ando
Ryoko
R
Fukano
Takashi
T
Hofkens
Johan
J
Miyawaki
Atsushi
A
D000199:Actins / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence; D017785:Photons; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins