Search for optimum two-photon excitation method with suppressed photobleaching of fluorescent proteins

2014 Fiscal Year Final Research Report

• Project/Area Number: 24560052
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied optics/Quantum optical engineering
• Research Institution: Tokyo University of Science
• Principal Investigator: SUDA AKIRA 東京理科大学, 理工学部, 教授 (80250108)
• Co-Investigator(Renkei-kenkyūsha): TAKAHASHI Hiroshi 東京理科大学, 理工学部, 助教 (70572709)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: 蛍光タンパク質 / 非線形分光 / 光褪色 / 二光子励起 / バイオイメージング

Outline of Final Research Achievements

Based on the measurement of the photobleaching action spectra of fluorescent proteins with nonlinear Fourier-transform spectroscopy, we found that the photobleaching occurs through excited-state absorption following two-photon excitation from the ground state. An unidentified dark state with a longer lifetime may be the initial state for excited-state absorption besides the triplet excited state. Then, the adaptive control method was applied to modulate the spectral amplitude and phase of excitation pulses maximizing the two-photon excited fluorescence while suppressing the photobleaching. As a result, the spectral amplitude was optimized to avoid the peak of the photobleaching spectra supporting the result of spectroscopic measurements.

Free Research Field

非線形光学