In single-molecule colocalization (“CoSMoS”) experiments, “the most challenging aspects of the technique are often not the preparation of the molecules or making the microscope observations, but in analyzing the resulting data. Data analysis has two primary challenges. First, one is studying inherently stochastic processes (thermally driven reactions of single molecules), so that analysis is inherently statistical. Second, the number of photons that can be emitted by a single fluorophore is limited by photobleaching, so images often have low signal-to-noise ratios, making discrimination of real signals from noise a challenge.”  In this article, Larry Friedman provides a step-by-step description the the procedures he has developed to reliably and efficiently analyze CoSMoS data.  While the examples discussed in the paper focus on analysis of transcription mechanisms, the methods should be applicable to CoSMoS studies on a broad range of biological systems.

Resources: Computer software that implements methods described in this article is available here.