Highlighted generation of fluorescence signals
using simultaneous two-color irradiation on Dronpa mutants
Ryoko Ando 1, Cristina Flors 2, Hideaki Mizuno 1, Johan Hofkens 2 and Atsushi Miyawaki 1*
1 Brain Science Institute of RIKEN
2 Katholieke Universiteit Leuven
* To whom correspondence should be addressed. E-mail: matsushi{at}brain.riken.jp.
Submitted on February 5, 2007
Revised on February 22, 2007
Accepted on 12 March 2007
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Abstract |
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Dronpa absorbs blue light and emits bright green fluorescence. It can also be converted by strong irradiation at 490 nm to a nonfluorescent state, which can then be switched back to the original emissive state with irradiation at 400 nm. Through semirandom mutagenesis studies, we have developed two mutants of Dronpa that show efficient photoswitching kinetics. Compared to Dronpa, the mutants can be turned off by blue light more efficiently. Thus, excitation with an argon laser line (488 nm) makes the mutants quickly become dark such that no substantial fluorescence signals can be observed. Excitation with a violet laser diode (405 nm) also produces no fluorescence signals. Simultaneous 488- and 405-nm irradiation, however, results in a rapid oscillation between the two states, thereby keeping the emissive state population large enough to produce sufficiently bright fluorescence signals.
Key Words:
fluorescence imaging, fluorescent protein, mutagenesis, photochromism
