具有非简并双光子激发的深度图像(会议报告)

Mu-Han Yang, Sanaz Sadegh, P. Saisan, Christopher G. L. Ferri, A. Devor, Y. Fainman
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摘要

在非简并双光子激发(ND-2PE)显微镜中,一个荧光团同时吸收两个不同能量的光子。我们对荧光蛋白和合成染料(如:eGFP, FITC等)连续变化两个光子的能量和数量,以创建荧光景观的二维地图。通过使用二维图中的最佳光子能量组合,我们发现当我们用增强的绿色荧光蛋白(eGFP)标记皮质神经元成像时,ND-2PE检测到的荧光图像亮度增加。应该注意的是,波长较长的光子在减少散射时将更深地穿透到组织中。此外,使用不同能量光子的非重叠空间模式可以显著减少大量低能光子的失焦荧光,通过适当选择高能量光子的数量,可以获得来自深层组织的ND-2PE荧光。实验上,我们策略性地置换了两束激光,直到它们到达样品平面,从而抑制了激发光束路径中不需要的背景。相比之下,这两个泵浦光束在焦点处很好地重叠,仍然产生足够数量的荧光光子用于检测。在我们的实验中,在红外光束的光程中使用光延迟线来实现时间对准。通过实验证明,与D-2PE相比,具有并行光束的ND-2PE在散射幻影中具有更好的信本比。研究了ND-2PE与D-2PE的激发体积,并确定其大小与D-2PE相当。
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Deep image with non-degenerated two-photon excitation (Conference Presentation)
In non-degenerate 2-photon excitation (ND-2PE) microscopy, a fluorophore simultaneously absorbs two photons of different energies. We performed a ND-2PE study of fluorescent proteins and synthetic dyes (eg.eGFP, FITC, and etc.) continuously varying energies and numbers of both photons to create two-dimensional map of fluorescence landscapes. By using the best photon energy combination from our two-dimensional map, we found an increase in detected fluorescent image brightness with ND-2PE as we imaged cortical neurons labeled with enhanced green fluorescent protein (eGFP). It should be noted that the photons corresponding to longer wavelength will penetrate deeper into the tissue at reduced scattering. Additionally, using non-overlapping spatial modes carrying the photons at different energies will significantly reduce out of focus fluorescence from the large number of low energy photons, and by a proper choice of the number of high energy photons the ND-2PE fluorescence can be obtained from deep tissue. Experimentally, we strategically displaced two laser beams until they reached the sample plane such that the unwanted background in the excitation beam path was suppressed. In contrast, these two pump beams were well overlapped at focus which still produced sufficient number fluorescence photons for detection. In our experiment, the temporal alignment was achieved with optical delay line in the optical path of IR beam. With this technique we demonstrated experimentally that ND-2PE with side-by-side beams provided a better signal to background ratio in the scattering phantom as compared with D-2PE. The excitation volume of ND-2PE with side-by-side beam was also investigated and determined to be comparable in size with that of the D-2PE.
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