How biomolecules behave in a squeeze - whether in response to confining walls or crowding agents, or inherent to molecular topologies - presents an open territory of exploration. We pioneer and apply single-molecule confinement microscopy to visualize and understand elusive interactions between biomolecules such as proteins and DNA. In this talk, I describe our investigations of the role of molecular topology, such as DNA supercoiling, in regulating the dynamic unwinding of specific target sites. In our experiments, the molecules are free to explore all possible configurations, and this has crucial influence over the dynamics we observe. Our goal is to achieve mechanistic insights into how gene regulation is influenced by DNA structure. In addition to studying topology-mediated DNA interactions, we confine and visualize a range of biomolecular systems that are challenging or impossible to access with other methods, including protein-protein and nanoparticle interactions that are relevant to emerging biotechnologies.