Monte Carlo simulation and maximum-likelihood analysis of single-molecule recycling in a nanochannel

Prolonged observation of a single molecule in solution using a confocal microscope is possible by flowing solution through a nanochannel and reversing the flow a fixed delay after each passage so that the molecule passes back and forth through the laser focus. In this paper, Monte Carlo simulations are used to provide insight on capabilities and limitations of the single-molecule recycling procedure. Various computational methods for using photon detection times to estimate the times of passage of the molecule through the laser focus, based on matched digital filters and maximum-likelihood (ML) analysis, are compared using simulations. A new ML-based methodology is developed for estimating the single molecule diffusivity, and the uncertainty in the estimate, from the variation in the intervals between times of passage. Simulations show that with recycling -200 times, it should be possible to resolve molecules with diffusivities that differ by a factor of -1.3, which is smaller than that resolvable in ligand-binding measurements by fluorescence correlation spectroscopy. Also, it is found that the mean number of times a molecule is recycled can be extended by adjusting the delay between flow reversals to accommodate the diffusional motion of statistical outliers. (c) 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement