Methods and applications of single-molecule fluorescence microscopy

The molecular mechanisms that support life depend on the fate of small populations of molecules. The functions of biomolecules, however, are traditionally characterised through bulk-phase biochemical methods that only report on ensemble averages. Such methods are not sensitive to population heterogeneity or short-lived reaction intermediates. Single-molecule methods that allow for the direct observation of individual molecules have significantly advanced our understanding of biology in the past two decades. The goal of the work described in this PhD thesis was the development and use of single-molecule techniques to answer biological questions about the process of DNA replication. DNA replication, the copying of the genome prior to cell division, is essential to all life forms. Previous single-molecule studies revealed unexpected and complex dynamics of this process. At the start of this thesis, I review the basic molecular principles of DNA replication across different organisms, with an emphasis on stochastic effects that have been revealed by single-molecule studies. Based on recent advances in the DNA-replication field, I hypothesise that dynamic interactions might be especially important to dealing with roadblocks of DNA replications, such as chemical lesions in the DNA or proteins bound to it.