Estimation of single-molecule blinking parameters using photon counting histogram

We have used computer simulations to explore the photon counting histogram (PCH) as a statistic for estimating kinetic parameters of a blinking immobilized fluorophore. Fluorescence trajectories made up of 500 on–off cycles allow recovery of the escape rates k on , k off with 20% error, when the bin size h is in an optimal range. The corresponding estimates of the intensities I on , I off improve with increasing resolution k on h , k off h of the on- and off-time, respectively. PCH analysis requires that the intensities I on , I off be well separated. The parameter estimates improve with increasing fluorescence trajectory length.