Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.
Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.
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DOI：
10.1038/nprot.2014.155
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发表时间：
2014-10
期刊：
影响因子：
14.8
通讯作者：
Hoskins AA
中科院分区：
文献类型：
--
作者：
Larson J;Kirk M;Drier EA;O'Brien W;MacKay JF;Friedman LJ;Hoskins AA
共定位单分子光谱（CoSMoS）已被证明是研究复杂细胞机器的组成、动力学和机制的有用方法。该技术的关键是能够同时监测多种蛋白质和/或核酸相互作用的能力。在这里，我们描述了一种构建CoSMoS微镜全反射荧光显微镜（mmTIRFM）的方案。科学显微镜的设计和建造通常需要许多定制组件和大量的时间承诺。在我们的协议中，我们通过实施商业上可用的显微镜平台来简化这一过程，该平台旨在容纳mmTIRFM所需的光学元件。mmTIRF系统消除了最终用户加工定制零件的需要，并便于光学校准。根据显微镜建造者的经验水平，这些节省的时间和以下协议可以使mmTIRF的建造在两个月内完成。
Colocalization Single Molecule Spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics, and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror Total Internal Reflection Fluorescence Microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a significant time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for a mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end-user and facilitates optical alignment. Depending on the experience-level of the microscope builder, these time-savings and the following protocol can enable mmTIRF construction to be completed within two months.