Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.
Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.
复制标题
DOI：
10.3390/biology10070571
复制
发表时间：
2021-06-23
期刊：
影响因子：
4.2
通讯作者：
Freudenthal BD
中科院分区：
文献类型：
--
作者：
Fairlamb MS;Whitaker AM;Bain FE;Spies M;Freudenthal BD
基于棱镜的单分子全内反射荧光显微镜是研究大分子动力学和相互作用的优秀工具。在这里，我们为使用市售组件的三色棱镜TIRF显微镜的设计、组装和操作提供了一个简单易用的指南，希望能帮助那些打算在他们的实验室实施TIRF成像技术的人。单分子全内反射荧光(TIRF)显微镜可以实时显示大分子动力学和复杂的组装。基于棱镜的TIRF显微镜操作相对简单，并且可以很容易地进行调制，以适应各种实验应用的需要。虽然在没有专家协助的情况下建造棱镜TIRF显微镜可能会带来巨大的挑战，但建造棱镜TIRF显微镜所需的组件相对来说是负担得起的，而且在一些指导下，组装工作可以由坚定的新手完成。在这里，我们为三色棱镜TIRF显微镜的设计、组装和操作提供了一个简单易操作的指南，该显微镜可用于研究大分子复合体，包括负责DNA修复、复制和转录的多组分蛋白质-DNA复合体。希望本文能对有志于实现单分子TIRF技术的实验室有所帮助，从而扩大该技术的应用范围。
Prism-based single-molecule total internal reflection fluorescence (prismTIRF) microscopes are excellent tools for studying macromolecular dynamics and interactions. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope using commercially available components with the hope of assisting those who aim to implement TIRF imaging techniques in their laboratory. Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein–DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.
登录
查看更多内容
DOI：
10.1021/acs.langmuir.5b02416
发表时间：
2015-09-22
期刊：
Langmuir : the ACS journal of surfaces and colloids
影响因子：
--
作者：
Gallardo IF;Pasupathy P;Brown M;Manhart CM;Neikirk DP;Alani E;Finkelstein IJ
通讯作者：
Finkelstein IJ
影响因子：
4.8
作者：
Bain, Fletcher E.;Wu, Colin G.;Spies, Maria
通讯作者：
Spies, Maria
DOI：
10.3791/50549
发表时间：
2014-04-24
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Chandradoss SD;Haagsma AC;Lee YK;Hwang JH;Nam JM;Joo C
通讯作者：
Joo C
影响因子：
16
作者：
Duderstadt, Karl E.;Geertsema, Hylkje J.;van Oijen, Antoine M.
通讯作者：
van Oijen, Antoine M.
影响因子：
4.8
作者：
Friedman, Larry J.;Gelles, Jeff
通讯作者：
Gelles, Jeff