Fig. 9.

Deviations from ideal SiMREPS behavior and suggested solutions. (A) Broadening of the N_b+d and/or τ distributions is frequently observed experimentally, resulting in less complete separation between specific (purple) and nonspecific (orange) binding distributions than theory predicts. This broadening can often be offset by a slight (e.g. < 2-fold) increase in the observation time to increase the number of FP binding events observed per target molecule. (B) If significant target dissociation occurs within the observation window, the N_b+d distribution will appear broader than expected. Increasing the CP’s affinity for the target may reduce the dissociation rate and tighten the distribution. (C) Secondary structure and other steric hindrance by adjacent sequence can broaden the apparent bound- or unbound-state lifetime distribution. In such cases, inclusion of one or more helper probes to block adjacent sequence can make the kinetics more homogeneous. (D) FPs may exhibit unwanted interactions with other sequences, particularly if other sequences are present at large surface densities or contain modifications such as LNAs that increase the affinity of base-pairing interactions. Even low-affinity interactions can interfere with measurements if they are numerous enough. In such cases, supplementing the imaging buffer with one or more blocker probes specific to the interfering sequence can improve the separation of signal and background peaks.