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

Abstract

Singlet molecular oxygen, O(2)(a(1)Delta(g)), has been detected from single neurons and HeLa cells in time-resolved optical experiments by its 1270 nm phosphorescence (a(1)Delta(g)--> X(3)Sigma(-)(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 H(2)O was replaced with D(2)O 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 H(2)O-containing cell, the lifetime of singlet oxygen is approximately 3 micros. 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.

Singlet Oxygen in a Cell: Spatially Dependent Lifetimes and Quenching Rate Constants

“…14,22,23 Time-resolved singlet oxygen phosphorescence experiments performed on D 2 O-incubated, TMPyP-containing cells yield an intracellular singlet oxygen lifetime of ~ 30-40 µs, irrespective of whether the data are recorded from the cytoplasm or the nucleus. [13][14][15][16] As expected based on the known solvent isotope effect (vide supra), this lifetime is progressively shortened for cells with an increasing ratio of intracellular H 2 O to D 2 O, and extrapolates to a value of ~ 3 µs in an H 2 O-containing cell. 15,16 It is now well established that "apparent" diffusion coefficients of small molecules inside a cell can be appreciably smaller than those in a homogeneous aqueous or hydrocarbon solvent.…”

“…[13][14][15][16] Specifically, for D 2 O-incubated cells, we repeatedly find that the lifetimes determined both in the cytoplasm and in the nucleus (~ 30-40 µs) are shorter than that for singlet oxygen in pure D 2 O (67 µs). These data point to a non-negligible k c [C] term, [13][14][15][16] as is indeed expected given that singlet oxygen can induce cell death. The quenching plots obtained using the NaN 3 equals the extracellular concentration in the incubating medium, the data in Figure 5 suggest that this intracellular quenching occurs at the diffusion-controlled limit.…”

“…HeLa cells were cultured and maintained using methods described in detail elsewhere. 16 For experiments in which singlet oxygen phosphorescence is detected from single Experiments were performed using cells exposed to an atmosphere of 100% oxygen, a condition that results in the most intense singlet oxygen phosphorescence signal. 15,28 Sensitizers were incorporated into the cells by incubating the cell in a medium that contained 10 µM of the dye, as previously described.…”

“…15,28 Sensitizers were incorporated into the cells by incubating the cell in a medium that contained 10 µM of the dye, as previously described. 16 For Chl, a 1 mM stock solution of the dye in DMSO was diluted to a final concentration of 10 µM in the incubation medium. The small amount of DMSO present in this case (1%) facilitated the incorporation of the hydrophobic Chl.…”

“…TMPyP is readily incorporated into a cell upon incubation of the cell in a medium that contains TMPyP. 16 Upon initial incorporation, TMPyP is first localized in lysosomes. 37,38 However, the dye eventually localizes in the nucleus (Figure 3a).…”