Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses.
The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. Our method will provide a powerful solution to various problems confronting researchers, such as the photobleaching of dyes in two-photon excitation microscopy.
Kawano
Hiroyuki
H
Laser Technology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. kawano@riken.go.jp
Nabekawa
Yasuo
Y
Suda
Akira
A
Oishi
Yu
Y
Mizuno
Hideaki
H
Miyawaki
Atsushi
A
Midorikawa
Katsumi
K
D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007834:Lasers; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D013997:Time Factors
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins