Measuring the lifetime of singlet oxygen in a single cell: addressing the issue of cell viability

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Abstract
Singlet molecular oxygen, O2(a1Δg), has been detected from single neurons and HeLa cells in time-resolved optical experiments by its 1270 nm phosphorescence (a1Δg → X3Σ– g) upon irradiation of a photosensitizer incorporated into the cell. The cells were maintained in a buffered medium and their viability was assessed by live/dead assays. To facilitate the detection of singlet oxygen, intracellular H2O was replaced with D2O by an osmotic de- and rehydration process. The effect of this insult on the cells was likewise assessed. The data indicate that, in the complicated transition from a “live” to “dead” cell, the majority of our cells have the metabolic activity and morphology characteristic of a live cell. Quenching experiments demonstrate that the singlet oxygen lifetime in our cells is principally determined by interactions with intracellular water and not by interactions with other cell constituents. The data indicate that in a viable, metabolically-functioning, and H2O-containing cell, the lifetime of singlet oxygen is ∼3 µs. This is consistent with our previous reports, and confirms that the singlet oxygen lifetime in a cell is much longer than hitherto believed. This implies that, in a cell, singlet oxygen is best characterized as a selective rather than reactive intermediate. This is important when considering roles played by singlet oxygen as a signaling agent and as a component in events that result in cell death. The data reported herein also demonstrate that spatially-resolved optical probes can be used to monitor selected events in the light-induced, singlet-oxygen-mediated death of a single cell.