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首页> 美国卫生研究院文献>Biophysical Journal >Excitation Spectra and Brightness Optimization of Two-Photon Excited Probes
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Excitation Spectra and Brightness Optimization of Two-Photon Excited Probes

机译：双光子激发探针的激发光谱和亮度优化

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摘要

Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced—resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.
机译：双光子探针激发数据通常表示为吸收截面或分子亮度（检测到的每个分子的荧光速率）。我们报告了基于光子相关光谱法的自动光谱系统，对各种有机和遗传编码探针的双光子分子亮度光谱进行了分析。可以在低激发强度下从分子亮度测量中提取双光子作用截面，而在更高的强度下测量峰值分子亮度（随着激发强度增加，最大分子亮度），在此强度下探针的光物理效应变得明显。在所有测试的染料家族中，这两个参数的光谱形状相似。峰值分子亮度光谱可以快速获得并且具有降低的实验复杂性，因此可以在确定用于双光子激发的最佳波长时，作为横截面光谱的一阶近似值，同时提供与探针光稳定性相关的其他信息。显示的数据应有助于多光子显微镜研究的探针选择和实验设计。此外，我们表明，通过添加无源脉冲分配器，可以减少非线性漂白，从而使荧光相关光谱中的荧光信号增强两倍。荧光信号的这种增加，以及观察到的作用截面和峰值亮度光谱的相似性，提示了用于双光子激发的更高阶的光漂白途径。

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