Journal article
Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules
Biology (Basel, Switzerland), Vol.10(7), 571
07/01/2021
DOI: 10.3390/biology10070571
PMCID: PMC8301196
PMID: 34201434
Abstract
Simple Summary Prism-based single-molecule total internal reflection fluorescence (prismTIRF) microscopes are excellent tools for studying macromolecular dynamics and interactions. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope using commercially available components with the hope of assisting those who aim to implement TIRF imaging techniques in their laboratory. 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.
Details
- Title: Subtitle
- Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules
- Creators
- Max S. Fairlamb - University of Kansas Medical CenterAmy M. Whitaker - University of Kansas Medical CenterFletcher E. Bain - University of IowaMaria Spies - University of IowaBret D. Freudenthal - University of Kansas Medical Center
- Resource Type
- Journal article
- Publication Details
- Biology (Basel, Switzerland), Vol.10(7), 571
- DOI
- 10.3390/biology10070571
- PMID
- 34201434
- PMCID
- PMC8301196
- NLM abbreviation
- Biology (Basel)
- ISSN
- 2079-7737
- eISSN
- 2079-7737
- Publisher
- Mdpi
- Number of pages
- 27
- Grant note
- Madison and Lila Self Graduate Fellowship R01-ES029203; R35GM128562; R35-GM131704; K99-ES031148 / National Institutes of Health; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Language
- English
- Date published
- 07/01/2021
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984293085702771
Metrics
15 Record Views