Improved timing and diffusivity measurement in single-molecule recycling in a nanochannel

Single-molecule recycling (SMR) in a nanochannel, in which a molecule in solution quickly passes through a focused laser beam and the solution flow is reversed after a set delay following each passage, provides an attractive alternative to feedback-driven trapping for prolonging the observation of a single molecule in a confocal microscope, as most of the time the molecule is in the dark, which extends the time before irreversible photobleaching and also gives time for recovery from photogenerated reversible dark states between passages. Guided by suggestions in previous SMR reports, we have utilized a National Instruments FPGA card and LabVIEW Realtime to implement 10 ns photon time-stamping, weighted sliding sum digital filtering, maximum-likelihood (ML) analysis of photon time-stamps, and real-time control of electrokinetic voltage in SMR experiments in order to improve the detection and timing of passages of the single molecule through the focused laser spot. We have developed a ML technique for measuring the diffusivity of the single molecule in the nanochannel, which uses a look-up table to update the probability density function of the diffusivity with each detected passage, thereby also providing confidence limits for the measurement. We use Monte Carlo simulations to examine prior experiments, validate the ML diffusivity measurement strategy, and evaluate choice of experimental parameters.