Singlet oxygen (¹O₂) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 μs in water. Whether the lifetime of ¹O₂ in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only ?220 nm during three lifetimes (?5%¹O₂ remaining), much shorter than cellular dimensions indicating that the primary reactions of ¹O₂ will be subcellularly localized near the site of ¹O₂ formation. This fact has raised the question of whether spatially resolved cellular responses to ¹O₂ occur, i.e. whether responses can be initiated by generation and reaction of ¹O₂ at a particular subcellular location that would not have been produced by ¹O₂ generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by ¹O₂ as a function of distance from the site of generation of ¹O₂. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo‐oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to ¹O₂ occur in cells.

Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy. A caveat is that the probes themselves should not perturb cellular responses and under normal conditions the dyes are generally non-toxic. However, researchers are increasingly using computerized time-lapse microscopy (CTLM), where cells stained with fluorescent dyes are often imaged frequently over a period of several days, to follow cellular responses in real time. Little is currently known about possible toxicity of fluorescent DNA dyes under CTLM conditions. In this study we demonstrate that the common live-cell DNA stain Hoechst 33342 can cause apoptosis under CTLM conditions. Although toxicity is evident at long times in the absence of imaging at high dye concentrations, phototoxicity from repeated excitation of the dye in the imaging process is dominant. We show that phototoxicity is a function of the product of light fluence and dye concentration, irrespective of irradiance, frequency and total number of scans. Thus, phototoxicity can be prevented by a combination of dye concentration and imaging procedure that is below this threshold. These quantitative data can be used as a guide to others performing time-lapse microscopy studies with this common live-cell DNA stain and serves as a caution for researchers when using other fluorescent stains under CTLM conditions.