| Summary: | 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.
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