Single-molecule methods are providing us with a growing insight into the nanoscopic world of single biomolecules. Quantitative tools based on force spectroscopy and fluorescence microscopy are now more regularly used to explore the physics of biomolecular processes. However, single-molecule experiments are typically limited to idealized in vitro conditions that are very different from the crowded situation in vivo. We aim to bridge this gap through a combination of approaches and the use of super-resolution microscopy methods. I will highlight our efforts to develop methods that can link idealized in vitro experiments with the realistic situation in vivo. Recent examples include imaging of DNA-associated molecular processes at high protein density and the use of multi-beam optical tweezers to control increasingly complex intermolecular interactions.