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bioRxiv · 10.1101/2021.05.10.443430

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

Fairlamb, M. S.·Whitaker, A. M.·Bain, F. E.·Spies, M.·Freudenthal, B. D.
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Abstract

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for 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 3-color prismTIRF microscope which can be utilized for the study 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 to a broader spectrum of scientific questions.

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Fairlamb, M. S., Whitaker, A. M., Bain, F. E., Spies, M., Freudenthal, B. D.. 2021-05-11. Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules. https://doi.org/10.1101/2021.05.10.443430

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