Four-wave mixing imaging to study protein entry and release in mammalian cells
Four-wave mixing imaging to study protein entry and release in mammalian cells
复制标题
四波混合成像研究哺乳动物细胞中蛋白质的进入和释放
DOI：
10.1016/j.drudis.2010.09.377
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发表时间：
2010
影响因子：
7.4
通讯作者：
Masia F
中科院分区：
文献类型：
--
作者：
Masia F
光学显微镜是追踪递送载体与哺乳动物细胞的结合、内化和亚细胞运输的强大工具。通过利用多光子过程，可以以固有的三维 (3D) 空间分辨率对亚细胞结构进行成像。多光子显微镜中常见的荧光标记包括有机荧光团（会遭受光漂白）和量子点（更耐光但含有细胞毒性元素（例如镉或铟））。金纳米粒子（GNP）在光稳定性和生物相容性方面是理想的光学标签，但发出微弱的荧光信号。我们开发了一种新颖的多光子显微镜技术，该技术利用了 GNP 与其表面等离子体共振的三阶非线性，称为四波混合 (FWM)。在成像性能方面，FWM显微镜具有优于单光子衍射极限的空间分辨率和光学切片能力。我们展示了金标记（尺寸小至 5 nm）的高对比度无背景成像以及对单颗粒水平的敏感性。我们还能够证明 GNP 与其表面结合蛋白的定向解离。这些结果为在治疗相关蛋白质受控释放到感兴趣的局部位点之前主动跟踪缀合纳米粒子铺平了道路。
Optical microscopy is a powerful tool for tracking the binding, internalisation and subcellular trafficking of delivery vectors to mammalian cells. By exploiting multiphoton processes, subcellular structures can be imaged with intrinsic three-dimensional (3D) spatial resolution. Common fluorescent labels in multiphoton microscopy include organic fluorophores, which suffer from photobleaching, and quantum dots which are more photostable but contain cytotoxic elements (such as Cd or In). Gold nanoparticles (GNPs) are ideal optical labels in terms of photostability and bio-compatibility, but emit weak fluorescent signal. We have developed a novel multiphoton microscopy technique that exploits the third-order nonlinearity called four-wave mixing (FWM) of GNPs in resonance with their surface plasmon. In terms of imaging performances, FWM microscopy features a spatial resolution better than the one-photon diffraction limit and optical sectioning capabilities. We show high-contrast background-free imaging of gold-labels (down to 5 nm size) and sensitivity to the single particle level. We are also able to demonstrate a directed dissociation of the GNP from bound proteins at their surface. These results pave the way for active tracking of conjugated nanoparticles, before the controlled release of therapeutically relevant proteins to a localised site of interest.