Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase.
Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase.
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DOI：
10.1007/s10895-015-1667-1
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发表时间：
2015-11
影响因子：
2.7
通讯作者：
Alexandrakis G
中科院分区：
文献类型：
--
作者：
Graham DJ;Tseng SF;Hsieh JT;Chen DJ;Alexandrakis G
在飞秒脉冲激发的双光子荧光蛋白质成像中，光漂白是一个关键的限制。我们介绍了测量EGFP光漂白与所用脉冲的光谱幅度和相位的关系。在800-950 nm的范围内，证实和测量了激发波长的强烈依赖性。利用光纤连续光源和脉冲整形技术，研究了具有不同光谱幅度和相位的宽带、15fs变换极限脉冲的光漂白。还研究了用于双光子成像应用的典型飞秒激光光源的窄带脉冲，>；150 fs变换极限，其光漂白与脉冲色散和带宽的关系。宽带脉冲的漂白速率主要由激发光的二次谐波光谱决定。另一方面，对于中心波长相近的窄带激发脉冲，漂白率的提高主要依赖于减小脉冲长度。提出了一个预测宽带脉冲相对漂白率的简单模型，并与实验数据进行了比较。
Photobleaching is a key limitation in two-photon imaging of fluorescent proteins with femtosecond pulsed excitation. We present measurements of the dependence of eGFP photobleaching on the spectral amplitude and phase of the pulses used. A strong dependence on the excitation wavelength was confirmed and measured over a 800–950 nm range. A fiber continuum light source and pulse shaping techniques were used to investigate photobleaching with broadband, 15 fs transform limited, pulses with differing spectral amplitude and phase. Narrow band pulses, >150 fs transform limited, typical of femtosecond laser sources used in two-photon imaging applications, were also investigated for their photobleaching dependence on pulse dispersion and bandwidth. The bleach rate for broadband pulses was found to be primarily determined by the second harmonic spectrum of the excitation light. On the other hand, for narrow band excitation pulses with similar center wavelengths improvement in bleach rate was found to be mostly dependent on reducing the pulse length. A simple model to predict the relative bleach rates for broadband pulses is presented and compared to the experimental data.