Optical detection of singlet oxygen from single cells

Abstract

The lowest excited electronic state of molecular oxygen, singlet molecular oxygen, O(2)(a (1)Delta(g)), is a reactive species involved in many chemical and biological processes. To better understand the roles played by singlet oxygen in biological systems, particularly at the sub-cellular level, optical tools have been developed to create and directly detect this transient state in time- and spatially-resolved experiments from single cells. Data obtained indicate that, contrary to common perception, this reactive species can be quite long-lived in a cell and, as such, can diffuse over appreciable distances including across the cell membrane into the extracellular environment. On one hand, these results demonstrate that the behavior of singlet oxygen in an intact cell can be significantly different from that inferred from model bulk studies. More generally, these results provide a new perspective for mechanistic studies of intra- and inter-cellular signaling and events that ultimately lead to photo-induced cell death.

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

“…[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.…”

“…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. 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.…”

“…It is also well-established that (i) changes in the concentration of dissolved oxygen only influence k T , not k rem (an exception occurs only in solvents where the inherent lifetime of singlet oxygen is extraordinarily long; e.g., CCl 4 or CS 2 ), and (ii) changes in the H/D isotopic composition of the surrounding medium only influence k rem , not k T . 2,15 In the present study, we used both direct triplet state measurements The insets show plots of the rate constant for singlet oxygen removal, k rem , against the concentration of NaN 3 (i.e., Eq. 2).…”

“…Although this phosphorescence is weak (φ ~ 10 -7 ), we have shown that it can nevertheless be detected from a single cell in both steady-state and time-resolved experiments upon irradiation of a sensitizer incorporated into the cell. [12][13][14][15][16] A key aspect of our work is that, using a focused laser beam, sensitizer excitation can be confined to small sub-cellular spatial domains. 15,17,18 Using this approach, it is now possible to provide unique insight into singlet-oxygen-mediated processes that occur in a cell.…”

“…Although we can impart spatial resolution to our experiments by irradiating the sensitizer in the cell with a focused laser, our present beam waist (diameter ~ 1 µm) 15,18 is still large relative to the structures that define intracellular heterogeneity. As such, we chose to work 6 with both hydrophobic (chlorin, Chl) and hydrophilic (TMPyP) sensitizers (Figure 1), expecting that these molecules would localize in different domains of the cell.…”