Construction of a three-color prism-based tirf microscope to study the interactions and dynamics of macromolecules

Max S. Fairlamb, Amy M. Whitaker, Fletcher E. Bain, Maria Spies, Bret D. Freudenthal

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

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.

Original languageEnglish
Article number571
JournalBiology
Volume10
Issue number7
DOIs
StatePublished - May 23 2021

Keywords

  • Construction guide
  • DNA repair complexes
  • Prism
  • Protein– DNA complexes
  • Single-molecule FRET
  • Single-molecule TIRF
  • SmFRET
  • Transcription complexes

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