Abstract
Direct observation of enzymes that work to promote nucleic acid metabolism is a powerful approach to understanding their biochemical and biological properties. Over several years, fluorescent optical microscopy has developed as a powerful tool for watching biological pathways as they occur in real time. Here we describe DNA curtains as an optical microscopy tool that combines engineering, biochemistry, and single molecule imaging to make direct observations of enzymes as they work on DNA in real time. We will provide a detailed methodology of this approach including information about the setup of a basic TIRF microscope, assembly of flow chambers for imaging, and the protocol for making DNA curtains. Our goal is to help the reader better understand the technical approaches to DNA curtains and to better understand the biochemical and biological applications of this approach.