单分子全内反射荧光(TIRF)显微镜允许大分子动力学和复杂组装的实时可视化。基于棱镜的TIRF显微镜(棱镜TIRF)操作相对简单,可以很容易地调整,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构建棱镜显微镜可能会带来重大挑战,构建棱镜TIRF显微镜所需的组件相对经济实惠,在一些指导下,组装可以由确定的新手完成。这里,我们提供了一个易于遵循的设计指南,装配,以及可用于研究大分子复合物的三色棱镜TIRF显微镜的操作,包括负责DNA修复的多组分蛋白质-DNA复合物,复制,和转录。我们希望这篇文章可以帮助那些渴望实施单分子TIRF技术的实验室,从而扩大了该技术的应用范围。
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.