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Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy
ACS Photonics ( IF 6.5 ) Pub Date : 2020-02-28 , DOI: 10.1021/acsphotonics.9b01749 Guy Malkinson 1 , Pierre Mahou 1 , Élodie Chaudan 2 , Thierry Gacoin 2 , Ali Y. Sonay 3 , Periklis Pantazis 3, 4 , Emmanuel Beaurepaire 1 , Willy Supatto 1
ACS Photonics ( IF 6.5 ) Pub Date : 2020-02-28 , DOI: 10.1021/acsphotonics.9b01749 Guy Malkinson 1 , Pierre Mahou 1 , Élodie Chaudan 2 , Thierry Gacoin 2 , Ali Y. Sonay 3 , Periklis Pantazis 3, 4 , Emmanuel Beaurepaire 1 , Willy Supatto 1
Affiliation
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Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO4 and BaTiO3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow.
中文翻译：
SHG纳米探针的多光子光片显微镜快速体内成像。
两光子光片显微镜（2P-SPIM）为活组织中的快速和深层荧光成像提供了优势的独特组合。除了荧光之外，检测相干信号（例如2P-SPIM中的二次谐波生成（SHG））还将打开进一步的成像机会。然而，光片显微镜法涉及照明和检测的正交配置，这质疑了检测相干信号的能力。实际上，微米级结构的相干散射主要发生在照明光束上。相比之下，点状光源（例如SHG纳米晶体）可以在多个方向上有效散射光，并可以使用光片显微镜的正交几何进行检测。这项研究调查了SHG光学显微镜（SHG-SPIM）对SHG纳米探针进行快速成像的适用性。理论和实验研究了SHG-SPIM 4和BaTiO 3纳米晶体。阐明了入射极化，检测数值孔径，纳米晶体旋转运动和二阶磁化率张量对称性对这种特定几何形状中SHG纳米探针可检测性的影响。建立了优化SHG-SPIM成像的指南，从而实现了将SHG和双光子激发荧光相结合的快速体内光片成像。最后，通过高达每秒180帧的SHG成像和血流中SHG纳米探针的单粒子跟踪，在活的斑马鱼胚胎中实现了微血管造影。
更新日期：2020-02-28
中文翻译：
SHG纳米探针的多光子光片显微镜快速体内成像。
两光子光片显微镜（2P-SPIM）为活组织中的快速和深层荧光成像提供了优势的独特组合。除了荧光之外，检测相干信号（例如2P-SPIM中的二次谐波生成（SHG））还将打开进一步的成像机会。然而，光片显微镜法涉及照明和检测的正交配置，这质疑了检测相干信号的能力。实际上，微米级结构的相干散射主要发生在照明光束上。相比之下，点状光源（例如SHG纳米晶体）可以在多个方向上有效散射光，并可以使用光片显微镜的正交几何进行检测。这项研究调查了SHG光学显微镜（SHG-SPIM）对SHG纳米探针进行快速成像的适用性。理论和实验研究了SHG-SPIM 4和BaTiO 3纳米晶体。阐明了入射极化，检测数值孔径，纳米晶体旋转运动和二阶磁化率张量对称性对这种特定几何形状中SHG纳米探针可检测性的影响。建立了优化SHG-SPIM成像的指南，从而实现了将SHG和双光子激发荧光相结合的快速体内光片成像。最后，通过高达每秒180帧的SHG成像和血流中SHG纳米探针的单粒子跟踪，在活的斑马鱼胚胎中实现了微血管造影。
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