Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

We first report on the development of new microscope means that reduce background contributions in fluores- cence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements al- low for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctua- tions in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.