Intracellular singlet oxygen photosensitizers: on the road to solving the problems of sensitizer degradation, bleaching and relocalization.

Selected singlet oxygen photosensitizers have been examined from the perspective of obtaining a molecule that is sufficiently stable under conditions currently employed to study singlet oxygen behavior in single mammalian cells. Reasonable predictions about intracellular sensitizer stability can be made based on solution phase experiments that approximate the intracellular environment (e.g.

Antioxidant β-carotene does not quench singlet oxygen in mammalian cells.

Carotenoids, and β-carotene in particular, are important natural antioxidants. Singlet oxygen, the lowest excited state of molecular oxygen, is an intermediate often involved in natural oxidation reactions. The fact that β-carotene efficiently quenches singlet oxygen in solution-phase systems is invariably invoked when explaining the biological antioxidative properties of β-carotene.

Irradiation- and sensitizer-dependent changes in the lifetime of intracellular singlet oxygen produced in a photosensitized process.

Singlet oxygen, O(2)(a(1)Δ(g)), was produced upon pulsed-laser irradiation of an intracellular photosensitizer and detected by its 1275 nm O(2)(a(1)Δ(g)) → O(2)(X(3)Σ(g)(-)) phosphorescence in time-resolved experiments using (1) individual mammalian cells on the stage of a microscope and (2) suspensions of mammalian cells in a 1 cm cuvette. Data were recorded using hydrophilic and, independently, hydrophobic sensitizers. The microscope-based single cell results are consistent with a model in which the behavior of singlet oxygen reflects the environment in which it is produced; nevertheless, the data also indicate that a significant fraction of a given singlet oxygen population readily crosses barriers between phase-separated intracellular domains.

Single cell responses to spatially controlled photosensitized production of extracellular singlet oxygen.

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 role of humic acid aggregation on the kinetics of photosensitized singlet oxygen production and decay.

The effect of humic acid (HA) aggregate formation on the photosensitized generation and subsequent quenching of singlet molecular oxygen O(2)(a(1)Δ(g)) was investigated. Time-resolved O(2)(a(1)Δ(g)) phosphorescence traces were obtained from (a) bulk samples of HA dispersions and (b) microscope-based experiments performed upon irradiation of a single HA aggregate. In the bulk experiments, the dependence of the O(2)(a(1)Δ(g)) lifetime on the HA concentration yields a critical concentration for the formation of micrometric HA aggregates of 0.

Singlet Oxygen Sensor Green®: photochemical behavior in solution and in a mammalian cell.

The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green(®) (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell.

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: achieving localized sub-cellular excitation in spatially-resolved experiments.

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation.

Photophysics of squaraine dyes: role of charge-transfer in singlet oxygen production and removal.

The unique optical properties of squaraines render these molecules useful for applications that range from xerography to photodynamic therapy. In this regard, squaraines derived from the condensation of nitrogen-based heterocycles and squaric acid have many promising attributes. Key solution-phase photophysical properties of six such squaraines have been characterized in this study.

Photosensitized production of singlet oxygen: spatially-resolved optical studies in single cells.

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.