Academic Journal
A Standardized Prism-Based TIRF Platform for Quantitative Single-Molecule Fluorescence Studies of Biomolecular Dynamics.
| Τίτλος: | A Standardized Prism-Based TIRF Platform for Quantitative Single-Molecule Fluorescence Studies of Biomolecular Dynamics. |
|---|---|
| Συγγραφείς: | Patra A; Department of Chemistry and Biochemistry, University of Mississippi, Oxford, MS 38677, USA., Melton L; Department of Chemistry and Biochemistry, University of Mississippi, Oxford, MS 38677, USA., Sawyer LS; Department of Chemistry and Biochemistry, University of Mississippi, Oxford, MS 38677, USA., King T; Department of Chemistry and Biochemistry, University of Mississippi, Oxford, MS 38677, USA., Ray S; Department of Chemistry and Biochemistry, University of Mississippi, Oxford, MS 38677, USA. |
| Πηγή: | Biosensors [Biosensors (Basel)] 2026 Jun 10; Vol. 16 (6). Date of Electronic Publication: 2026 Jun 10. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: MDPI Pub Country of Publication: Switzerland NLM ID: 101609191 Publication Model: Electronic Cited Medium: Internet ISSN: 2079-6374 (Electronic) Linking ISSN: 20796374 NLM ISO Abbreviation: Biosensors (Basel) Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Basel, Switzerland : MDPI Pub. |
| Ιατρικοί όροι (MeSH): | Fluorescence Resonance Energy Transfer* , Single Molecule Imaging*, Microscopy, Fluorescence/methods ; Signal-To-Noise Ratio ; Calibration ; Fluorescence |
| Περίληψη: | Single-molecule Förster resonance energy transfer (smFRET) enables direct measurement of nanoscale conformational dynamics and heterogeneity in biomolecules, but quantitative interpretation of smFRET data critically depends on well-controlled excitation geometry, low background fluorescence, robust calibration, and reproducible data-analysis workflows. Prism-based total internal reflection fluorescence (pTIRF) microscopy provides important advantages for such measurements by physically separating excitation and emission paths and generating a highly confined evanescent field, yet practical guidance for implementing reproducible, quantitative pTIRF systems remains fragmented. Here we present a comprehensive, standardized framework for the design, alignment, calibration, validation, and operation of a prism-based TIRF microscope optimized for single-molecule fluorescence measurements. We describe the complete optical architecture for dual-color excitation and detection, establish alignment invariants that ensure reproducible evanescent excitation and stable donor-acceptor channel registration, and detail surface preparation, flow control, and photostabilization strategies required for reliable long-term imaging. Quantitative benchmarking protocols are introduced to evaluate signal-to-noise ratio, photobleaching kinetics, and spectral crosstalk, providing objective criteria for defining optimal operating conditions and instrument performance limits. Finally, we integrate these experimental procedures with an end-to-end single-molecule data-analysis workflow encompassing channel registration, automated and manual trajectory selection, FRET calculation, and kinetic analysis using hidden Markov modeling. The utility of the platform is demonstrated through smFRET measurements of conformational dynamics in a model nucleic acid system. Together, this work provides a reproducible and accessible methodology for implementing prism-based TIRF microscopy as a robust quantitative platform for single-molecule fluorescence studies across a wide range of biomolecular systems. |
| Grant Information: | Startup University of Mississippi |
| Contributed Indexing: | Keywords: donor leakage; evanescent wave; flow-cell preparation; microscope construction; optical alignment; photobleaching statistics; prism-based TIRF (pTIR); prism-type total internal reflection fluorescence microscopy; quantitative performance benchmarks; signal-to-noise ratio; single-molecule data analysis; single-molecule fluorescence; smFRET; surface immobilization |
| Entry Date(s): | Date Created: 20260625 Date Completed: 20260625 Latest Revision: 20260726 |
| Update Code: | 20260726 |
| PubMed Central ID: | PMC13296891 |
| DOI: | 10.3390/bios16060331 |
| PMID: | 42345887 |
| Βάση Δεδομένων: | MEDLINE |
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