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Updated: Jul 16, 2026
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Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
Published on: February 7, 2021
Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood
Joshua B Edel1, John S Eid, Amit Meller
1Department of Physics and Biomedical Engineering, Boston University, 44 Cummington Street, Boston, Massachusetts 02215, USA.
The Journal of Physical Chemistry. B
|March 29, 2007
Summary
We developed a new method to accurately measure single DNA molecule fluorescence lifetimes, even with low photon counts. This technique corrects for background noise, improving the reliability of fluorescence lifetime measurements.
Area of Science:
- Biophysics
- Molecular Biology
- Spectroscopy
Background:
- Single-molecule fluorescence spectroscopy is crucial for understanding DNA dynamics.
- Accurate fluorescence lifetime measurements are essential for FRET-based studies.
- Background fluorescence and instrument response can complicate data analysis.
Purpose of the Study:
- To develop a robust method for measuring single-molecule fluorescence lifetime trajectories of DNA.
- To improve the accuracy of fluorescence lifetime extraction from low photon count data.
- To address challenges posed by background fluorescence in single-molecule measurements.
Main Methods:
- Utilized time-tagged single-photon counting and scanning confocal microscopy.
- Immobilized double-stranded DNA with tetramethylrhodamine and Cy5 FRET pair.
- Developed a modified maximum likelihood estimator (MLE) algorithm.
Main Results:
- Successfully extracted fluorescent lifetimes from decays with as few as 20 photons.
- Demonstrated the robustness of the MLE algorithm in compensating for background and instrument response.
- Showed that extracted lifetimes are highly dependent on background fluorescence levels.
Conclusions:
- The modified MLE algorithm enables reliable extraction of true fluorescence lifetime trajectories from single fluorophores.
- Appropriate correction factors are necessary for accurate lifetime determination in the presence of background.
- This method enhances the precision of single-molecule DNA studies using FRET.
