Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging
Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging
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DOI：
10.1038/nprot.2016.022
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发表时间：
2016-04-01
期刊：
影响因子：
14.8
通讯作者：
Kukura, Philipp
中科院分区：
文献类型：
--
作者：
Arroyo, Jaime Ortega;Cole, Daniel;Kukura, Philipp
干涉散射显微镜（iSCAT）是一种基于光散射的成像模式，它提供了成像速度和精度的独特组合，用于跟踪纳米级标签，并实现了低至单分子水平的无标签光学传感。与荧光相反，iSCAT不受与染料光化学和荧光物理学相关的限制，也不需要荧光标记。在这里，我们提出了一个协议，用于构建iSCAT显微镜从市售的光学元件，并证明其兼容性，同时操作单分子，客观型，全内反射荧光显微镜。鉴于光学和显微镜的中级经验，例如对激光束转向和光学组件的研究生水平的熟悉，该方案可以在2周的时间内完成。
Interferometric scattering microscopy (iSCAT) is a light scattering-based imaging modality that offers a unique combination of imaging speed and precision for tracking nanoscopic labels and enables label-free optical sensing down to the single-molecule level. In contrast to fluorescence, iSCAT does not suffer from limitations associated with dye photochemistry and photophysics, or the requirement for fluorescent labeling. Here we present a protocol for constructing an iSCAT microscope from commercially available optical components and demonstrate its compatibility with simultaneously operating single-molecule, objective-type, total internal reflection fluorescence microscopy. Given an intermediate level of experience with optics and microscopy, for instance graduate-level familiarity with laser beam steering and optical components, this protocol can be completed in a time frame of 2 weeks.