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Quantification of Rare Single-Molecule Species Based on Fluorescence Lifetime
Cong Liu1, Ajay Rastogi1, Hsin-Chih Yeh1
1Department of Biomedical Engineering, Cockrell School of Engineering, University of Texas at Austin , Austin, Texas 78712, United States.
Analytical Chemistry
|April 12, 2017
Summary
Accurate quantification of rare molecules using single-molecule tracking requires millions of data points, even with high identification accuracy. This study reveals the significant challenges in precise molecular counting without amplification.
Area of Science:
- Biophysics
- Analytical Chemistry
- Molecular Biology
Background:
- Single-molecule tracking (SMT) combined with fluorescence lifetime analysis offers direct molecular quantification in solution.
- Accurate quantification of rare molecular species using SMT remains challenging due to uncertainties in molecular identification accuracy and the number of required tracks.
Purpose of the Study:
- To determine the molecular identification accuracy and number of single-molecule tracks necessary for accurate quantification of rare molecular species.
- To assess the fundamental challenges in single-molecule identification and quantification without amplification.
Main Methods:
- Computational analysis based on experimentally obtained single-molecule fluorescence lifetime data.
- Application of an unbiased ratio estimator to quantify rare molecular species.
Main Results:
- Even with a molecular identification accuracy of 0.99999, approximately 1.8 million single-molecule tracks are necessary.
- Achieving a 95% confidence level with a relative error less than ±5% for rare-species quantification demands a substantial number of tracks.
Conclusions:
- Accurate quantification of rare molecular species using SMT without amplification is fundamentally challenging.
- A high number of single-molecule tracks, even with high identification accuracy, is required to overcome inherent uncertainties.