Brightness-gated two-color coincidence detection unravels two distinct mechanisms in bacterial protein translation initiation

Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Here, we introduce a brightness-gated TCCD which overcomes this limitation and benchmark our approach with two custom-made calibration samples. Applied to a cell-free protein synthesis assay, brightness-gated TCCD unraveled a previously disregarded mode of translation initiation in bacteria. Henning Höfig et al. present a brightness-gated two-color coincidence detection method that overcomes the limitations of confocal detection. The method adds a selection criterion for the brightness of single fluorescence bursts, substantially improving the counting accuracy for single fluorophores.