Excitation Spectra and Brightness Optimization of Two-Photon Excited Probes
Excitation Spectra and Brightness Optimization of Two-Photon Excited Probes
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DOI：
10.1016/j.bpj.2011.12.056
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发表时间：
2012-02-22
影响因子：
3.4
通讯作者：
Harris, Timothy D.
中科院分区：
文献类型：
--
作者：
Muetze, Joerg;Iyer, Vijay;Harris, Timothy D.
两光子探针激发数据通常表示为吸收横截面或分子亮度（每个分子检测到的荧光速率）。我们报告了基于荧光相关光谱学的自动光谱系统的一组有机和遗传编码探针的两光子分子亮度光谱。在低激发强度下，可以从分子亮度测量中提取两光子作用横截面，而在较高的强度下，测量峰值分子亮度（最大的分子亮度随增加激发强度的增加），在较高的强度下，探针光物理效应变得显着。在所有测试的染料家族中，这两个参数的光谱形状相似。因此，可以快速获得峰值分子亮度光谱，并以降低的实验复杂性获得，因此可以用作横截面光谱的一阶近似，以确定两光子激发的最佳波长，同时提供与探针光稳定性有关的其他信息。所示的数据应有助于探针选择和实验设计，以进行多光子显微镜研究。此外，我们表明，通过添加被动脉冲分离器，在荧光相关光谱中的荧光信号增强荧光信号时，非线性漂白可以减少两个因子。荧光信号的增加，以及观察到的动作横截面和峰亮度光谱的相似之处，提示了两光子激发的高阶光漂白途径。
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.