Quantification of Rare Single-Molecule Species Based on Fluorescence Lifetime.

Single-molecule tracking combined with fluorescence lifetime analysis can be a powerful tool for direct molecular quantification in solution. However, it is not clear what molecular identification accuracy and how many single-molecule tracks are required to achieve an accurate quantification of rare molecular species. Here we carry out calculations to answer these questions, based on experimentally obtained single-molecule lifetime data and an unbiased ratio estimator. Our results indicate that even at the molecular identification accuracy of 0.99999, 1.8 million tracks are still required in order to achieve 95% confidence level in rare-species quantification with relative error less than ±5%. Our work highlights the fundamental challenges that we are facing in accurate single-molecule identification and quantification without amplification.