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Combined Scattering, Interferometric, and Fluorescence Oblique Illumination for Live Cell Nanoscale Imaging
ACS Photonics ( IF 7 ) Pub Date : 2022-11-14 , DOI: 10.1021/acsphotonics.2c01143
Yujie Zheng, Yean Jin Lim, Hanqi Lin, Tienan Xu, Carmen Longbottom, Viviane Delghingaro-Augusto, Yee Lin Thong, Christopher R. Parish, Elizabeth E. Gardiner, Woei Ming Lee

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label-free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structures. A combined imaging system would permit obtaining quantitative 3D spatial temporal details of individual cellular components. In this paper, we empirically determined optimal azimuthal scanning angles of rotating beams to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, bright-field (BF) and dark-field (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high-inclined laminated oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering, and fluorescence imaging enable (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of more than ∼1000 platelets suggest that serum albumin (bovine) is necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we identified new asynchronous membrane fluctuations between the leading and rear edges of a migrating platelet. We further demonstrated that interferometric imaging permits the quantification of mitochondrial dynamics on human lung microvascular endothelial cells. Our data suggests that axial displacement of mitochondria is minimal when it is closer to the nucleus or the leading edge of a cell membrane. Taken together, this combined nanoscopy platform helps to quantify multiple spatial temporal events of a migrating cell that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

中文翻译:

用于活细胞纳米级成像的组合散射、干涉和荧光倾斜照明

要使用活细胞成像工具确定细胞迁移的分子和/或机械基础,有必要同时关联多个 3D 时空事件。荧光纳米显微镜和无标记纳米级成像可以通过提供亚细胞结构的分子特异性和结构动力学相互补充。组合成像系统将允许获得单个细胞成分的定量 3D 时空细节。在本文中,我们凭经验确定了旋转光束的最佳方位角扫描角度,以实现同时且无标记的纳米级和荧光成像。这里的无标记纳米级成像是指干涉、明场(BF)和暗场(DF)旋转相干散射(ROCS)显微镜,而荧光是指高倾斜层压斜(HiLO)和全内反射荧光(TIRF)成像。干涉、散射和荧光成像的综合能力使 (1) 分子目标(底物或细胞器)的识别,(2)3D 细胞形态动力学的量化,以及(3)在 3D 中跟踪细胞内细胞器。然后使用这种组合成像工具来表征迁移的血小板和内皮细胞,这两者对感染和伤口愈合过程都至关重要。超过 1000 个血小板的综合成像结果表明,血清白蛋白(牛)是血小板迁移和清除纤维蛋白/纤维蛋白原所必需的。此外,我们在迁移的血小板的前缘和后缘之间发现了新的异步膜波动。我们进一步证明,干涉成像允许对人肺微血管内皮细胞的线粒体动力学进行量化。我们的数据表明,当线粒体靠近细胞核或细胞膜前缘时,其轴向位移最小。总而言之,这种组合的纳米显微镜平台有助于量化迁移细胞的多个时空事件,这无疑将为新的定量相关纳米级活细胞成像开辟道路。
更新日期:2022-11-14
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