Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual proteins and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these proteins function in concert with other proteins in large complexes, so an important direction is the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. I will discuss the development of such tools to study DNA replication. In particular, I will focus on the role of micromanipulation techniques, microfluidic approaches, and imaging methods in such single-molecule studies. Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved.