Raman and SERS microscopy for molecular imaging of live cells
Raman and SERS microscopy for molecular imaging of live cells
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DOI：
10.1038/nprot.2013.030
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发表时间：
2013-04-01
期刊：
影响因子：
14.8
通讯作者：
Fujita, Katsumasa
中科院分区：
文献类型：
--
作者：
Palonpon, Almar F.;Ando, Jun;Fujita, Katsumasa
拉曼显微镜是一种很有前途的技术，用于可视化细胞中分子的分布。使用拉曼显微镜的活细胞成像的挑战是由于弱的拉曼信号导致的长成像时间。在这里，我们提出了一个协议，用于构建和使用拉曼显微镜配备了狭缝扫描激发和检测系统和激光转向和纳米粒子跟踪系统。狭缝扫描允许具有高时间和空间分辨率的拉曼成像，而激光束转向系统允许使用金纳米颗粒的动态表面增强拉曼成像。这两个功能都可以绘制活细胞中分子的分布图，并可视化细胞转运途径。此外，它的效用可以扩展到小分子成像，通过使用微小的拉曼活性标签，如炔。例如，细胞中的DNA合成可以通过检测5-乙炔基-2 '-脱氧尿苷（EdU）（一种具有炔部分的脱氧尿苷衍生物）来可视化。我们描述了光学，硬件和软件来构建的拉曼显微镜，并讨论了条件和参数参与活细胞成像。整个系统可以在大约8小时内建成。
Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and a laser steering and nanoparticle-tracking system. Slit scanning allows Raman imaging with high temporal and spatial resolution, whereas the laser beam steering system enables dynamic surface-enhanced Raman imaging using gold nanoparticles. Both features enable mapping of the distributions of molecules in live cells and visualization of cellular transport pathways. Furthermore, its utility can be expanded to small-molecule imaging by using tiny Raman-active tags such as alkyne. For example, DNA synthesis in a cell can be visualized by detecting 5-ethynyl-2'-deoxyuridine (EdU), a deoxyuridine derivative with an alkyne moiety. We describe the optics, hardware and software to construct the Raman microscope, and discuss the conditions and parameters involved in live-cell imaging. The whole system can be built in similar to 8 h.