In this feature article, we focus on one ROS in particular: singlet oxygen, O2(a1Δg). We summarize our recent efforts to selectively produce singlet oxygen in sub-cellular, spatially-resolved experiments performed on single mammalian cells. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to initially create a localized femtoliter volume of singlet oxygen, (2) protein-encapsulated photosensitizers that can be localized in cells using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments. We also provide a brief overview of our recent efforts to monitor singlet oxygen in cells (e.g., direct time-resolved optical detection, fluorescent probes) and to monitor the cell’s response to singlet oxygen (e.g., the use of rapid super-resolution microscopy). In all cases, we discuss the advantages and disadvantages of that particular approach/tool.