Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. I will present our developments of single-molecule imaging and microfluidics techniques to investigate the molecular mechanisms of DNA repair and mutagenesis in single Escherichia coli cells. With these tools, we were able to follow the real-time dynamics of repair and mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating mutagenesis to the chronology of the underlying molecular processes. By avoiding population averaging, we discovered that cell-to-cell variation in mutagenesis correlated with heterogeneity in the expression of alternative responses to DNA damage.