Singlet molecular oxygen, O(2)(a(1)Delta(g)), can be created in photosensitized experiments with sub-cellular spatial resolution in a single cell. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a(1)Delta(g)--> X(3)Sigma). Cellular responses to the creation of singlet oxygen can be monitored using viability assays. Time- and spatially-resolved optical measurements of both singlet oxygen and its precursor, the excited state sensitizer, reflect the complex and dynamic morphology of the cell. These experiments help elucidate photoinduced, oxygen-dependent events that compromise cell function and ultimately lead to cell death. In this perspective, recent work on the photosensitized production and detection of singlet oxygen in single cells is summarized, highlighting the advantages and current limitations of this unique experimental approach to study an old problem.

The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed post-irradiation “waiting period” before necrosis became apparent depended on (a) the distance between the cell membrane and the domain irradiated, (b) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced, and (c) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach of using extracellularly-generated singlet oxygen to induce cell death can provide a solution to a problem that often limits mechanistic studies of intracellularly photosensitized cell death: it can be difficult to quantify the effective light dose, and hence singlet oxygen concentration, when using an intracellular photosensitizer.

Oxygen diffusion coefficients have been determined in ethanol-swollen poly(vinyl alcohol), PVA, gels using a technique wherein oxygen sorption is optically monitored using singlet oxygen phosphorescence. Data were recorded as a function of the extent to which the PVA chains are chemically cross-linked using glutaraldehyde. Contrary to conventional expectation, the diffusion coefficients obtained increase with an increase in the extent of cross-linking. This observation is interpreted in terms of a cross-link-dependent increase in the microscopic heterogeneity of the polymer wherein dense cross-linked domains coexist with more fluid domains. It is expected that, in the latter domains, segmental motions of the macromolecule that facilitate oxygen diffusion are more readily achieved. This model of cross-link-dependent heterogeneity is supported by the results of small-angle X-ray scattering experiments. Among other things, the data reported herein provide an informative foundation for studies of small molecule diffusion in biological cells, particularly during photoinduced cell death where the hydrogel-like nature of the cell can change due to cross-linking reactions.