PT  - JOURNAL ARTICLE
AU  - Max S. Fairlamb
AU  - Amy M. Whitaker
AU  - Fletcher E. Bain
AU  - Maria Spies
AU  - Bret D. Freudenthal
TI  - Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules
AID  - 10.1101/2021.05.10.443430
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.10.443430
4099  - http://biorxiv.org/content/early/2021/05/11/2021.05.10.443430.short
4100  - http://biorxiv.org/content/early/2021/05/11/2021.05.10.443430.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.