Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-06-01
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| Series: | Biology |
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| Online Access: | https://www.mdpi.com/2079-7737/10/7/571 |
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| author | Max S. Fairlamb Amy M. Whitaker Fletcher E. Bain Maria Spies Bret D. Freudenthal |
| author_facet | Max S. Fairlamb Amy M. Whitaker Fletcher E. Bain Maria Spies Bret D. Freudenthal |
| author_sort | Max S. Fairlamb |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T10:10:15Z |
| format | Article |
| id | doaj.art-f1215662bead45bf841227af13a76c12 |
| institution | Directory Open Access Journal |
| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-10T10:10:15Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biology |
| spelling | doaj.art-f1215662bead45bf841227af13a76c122023-11-22T01:18:12ZengMDPI AGBiology2079-77372021-06-0110757110.3390/biology10070571Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of MacromoleculesMax S. Fairlamb0Amy M. Whitaker1Fletcher E. Bain2Maria Spies3Bret D. Freudenthal4Department of Biochemistry and Molecular Biology and Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USADepartment of Biochemistry and Molecular Biology and Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USADepartment of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, 51 Newton Road, Iowa City, IA 52242, USADepartment of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, 51 Newton Road, Iowa City, IA 52242, USADepartment of Biochemistry and Molecular Biology and Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USASingle-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.https://www.mdpi.com/2079-7737/10/7/571single-molecule TIRFprismsingle-molecule FRETsmFRETconstruction guideprotein–DNA complexes |
| spellingShingle | Max S. Fairlamb Amy M. Whitaker Fletcher E. Bain Maria Spies Bret D. Freudenthal Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules Biology single-molecule TIRF prism single-molecule FRET smFRET construction guide protein–DNA complexes |
| title | Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules |
| title_full | Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules |
| title_fullStr | Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules |
| title_full_unstemmed | Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules |
| title_short | Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules |
| title_sort | construction of a three color prism based tirf microscope to study the interactions and dynamics of macromolecules |
| topic | single-molecule TIRF prism single-molecule FRET smFRET construction guide protein–DNA complexes |
| url | https://www.mdpi.com/2079-7737/10/7/571 |
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