Exerting better control and specificity with singlet oxygen experiments in live mammalian cells

Singlet molecular oxygen, O₂(a¹Δ_g), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O₂(a¹Δ_g) in this regard is acknowledged, an understanding of the targets and mechanisms of O₂(a¹Δ_g) action is inadequate. Thus, methods that better facilitate studies of O₂(a¹Δ_g) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O₂(a¹Δ_g) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O₂(a¹Δ_g) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O₂(a¹Δ_g) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O₂(a¹Δ_g) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O₂(a¹Δ_g) at the expense of other ROS. We also comment on our recent efforts to monitor O₂(a¹Δ_g) in cells and to monitor the cell's response to O₂(a¹Δ_g).