Fairlamb Max S, Whitaker Amy M, Bain Fletcher E, Spies Maria, Freudenthal Bret D
Department of Biochemistry and Molecular Biology and Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, 51 Newton Road, Iowa City, IA 52242, USA.
Biology (Basel). 2021 Jun 23;10(7):571. doi: 10.3390/biology10070571.
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.
单分子全内反射荧光（TIRF）显微镜能够实时观察大分子动力学和复杂组装过程。基于棱镜的TIRF显微镜（prismTIRF）操作相对简单，并且可以轻松调节以适应各种实验应用的需求。虽然在没有专家协助的情况下构建prismTIRF显微镜可能是一项重大挑战，但构建该显微镜所需的组件相对价格合理，并且在一些指导下，有决心的新手也可以完成组装。在这里，我们提供了一个易于遵循的指南，用于设计、组装和操作三色prismTIRF显微镜，该显微镜可用于研究大分子复合物，包括负责DNA修复、复制和转录的多组分蛋白质-DNA复合物。我们希望本文能够帮助有志于应用单分子TIRF技术的实验室，从而扩大该技术的应用范围。
