Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli, we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions.