Abstract
Simple SummaryPrism-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.
Highlights
Prism-based single-molecule total internal reflection fluorescence microscopy is becoming ever-more popular due to its wide variety of applications
Multiple publications are available which outline the various capabilities of prismTIRF microscopes [1,2], the rationale behind fluorophore selection [3,4,5], the general assembly of a two-color prismTIRF microscope [6,7,8], and the basics of how to perform such experiments [3,9,10,11,12]
Here we provide an easy-to-follow detailed guide for designing, assembling, and operating a three-color prismTIRF microscope which can be utilized for the study of biologically relevant macromolecular assemblies, including protein–DNA complexes
Summary
Prism-based single-molecule total internal reflection fluorescence (prismTIRF) microscopy is becoming ever-more popular due to its wide variety of applications. Multiple publications are available which outline the various capabilities of prismTIRF microscopes [1,2], the rationale behind fluorophore selection [3,4,5], the general assembly of a two-color prismTIRF microscope [6,7,8], and the basics of how to perform such experiments [3,9,10,11,12]. Even with the currently available guides, building and operating a prismTIRF microscope without expert assistance can be a challenge With this in mind, here we provide an easy-to-follow detailed guide for designing, assembling, and operating a three-color prismTIRF microscope which can be utilized for the study of biologically relevant macromolecular assemblies, including protein–DNA complexes. The fourth section (Application) provides pertinent examples of how TIRF microscopes have been utilized to study DNA replication, DNA repair, and DNA transcription
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