Time-resolved Singlet Oxygen Phosphorescence Measurements from Photosensitized Experiments in Single Cells: Effects of Oxygen Diffusion and Oxygen Concentration

Photochemistry and Photobiology, Sep/Oct 2008 by Hatz, Sonja, Poulsen, Lars, Ogilby, Peter R

ABSTRACT

Time-resolved singlet oxygen, O2(a^sup 1^Δ^sub g^), phosphorescence experiments have been performed in single cells upon pulsed laser irradiation of a photosensitizer incorporated into the cell. Data recorded as a function of the partial pressure of ambient oxygen to which the cell is exposed reflect apparent values for the intracellular oxygen diffusion coefficient and intracellular oxygen concentration that are smaller than those found in neat H2O. This conclusion is supported by O2(a^sup 1^Δ^sub g^) phosphorescence data and sensitizer triplet state absorption data recorded in control experiments on sucrose solutions with different viscosities. We recently demonstrated that the intracellular lifetime of O2(a^sup 1^Δ^sub g^) is comparatively long (~3 µs) and does not differ significantly from that in neat H2O (-3.5 µs). Despite this long lifetime, however, our estimate of an apparent intracellular oxygen diffusion coefficient in the range ~2-4 x 10^sup -6^ cm^sup 2^ s^sup -1^ means that the spatial domain of intracellular O2(a^sup 1^Δ^sub g^) activity will likely have a spherical radius of ~100 nm. This latter point helps reconcile seeming inconsistencies between our direct O2(a^sup 1^Δ^sub g^) lifetime data and results obtained from independent photobleaching experiments that show a limited translational diffusion distance for O2(a^sup 1^Δ^sub g^) within a cell.

INTRODUCTION

Singlet oxygen, O2(a^sup 1^Δ^sub g^), is the lowest excited electronic state of molecular oxygen. It has a unique chemistry that results in the oxygenation of a host of organic molecules (1). These reactions can be particularly important in biological systems. For example, O2(a^sup 1^Δ^sub g^) is involved in events that result in both necrotic and apoptotic cell death where, through its specific reactions, it can have roles that range from participating in signaling mechanisms to the direct and adverse oxygenation of a key cellular component (2).

A common and convenient method by which O2(a^sup 1^Δ^sub g^) can be produced is photosensitization (Fig. 1) (3). In this process, a molecule (the so-called sensitizer) absorbs light to populate the lowest excited singlet state, S^sub 1^. Although O2(a^sup 1^Δ^sub g^) can be produced upon quenching of S^sub 1^ by ground state oxygen, O2(X^sub 3^Σ^sub g^^sup -^), the most efficient sensitizers readily undergo intersystem crossing to produce a longer lived triplet state which is more effectively quenched by O2(X^sup 3^Σ^sub g^^sup -^) to produce O2(a^sup 1^Δ^sub g^). For some sensitizers, this process of intersystem crossing is promoted by O3(X^sup 3^Σ^sub g^^sup -^) which can have consequences in photosensitized O2(a^sup 1^Δ^sub g^) experiments performed as a function of the ambient oxygen partial pressure (4).

The most unambiguous way to monitor O2(a^sup 1^Δ^sub g^) is arguably in a direct, time-resolved spectroscopic experiment. Indeed, over the past ~25 years, the O2(a^sup 1^Δ^sub g^) [arrow right] O2(X^sup 3^Σ^sub g^^sup -^) phosphorescence at 1270 nm has proved to be a most valuable tool (3). We have recently shown that this phosphorescence can be detected in photosensitized experiments from single cells (5-9).

In performing these experiments on single cells, we noted that the intensity of our O2(a^sup 1^Δ^sub g^) phosphorescence signal was quite sensitive to the oxygen partial pressure to which the cell was exposed. Specifically, we observed an appreciable increase in the signal intensity as the ambient atmosphere was changed from air to oxygen. In contrast, using the same sensitizer, corresponding experiments performed in bulk aqueous solutions revealed very little change in the intensity of the O2(a^sup 1^Δ^sub g^) signal as the ambient atmosphere was changed from air to oxygen.

The effect of the ambient oxygen partial pressure on both the time evolution and intensity of O2(a^sup 1^Δ^sub g^) phosphorescence signals in photosensitized experiments has been examined in detail for a variety of sensitizers in homogeneous liquid solutions (4,10,11). The data reflect the expected effect that, at low oxygen concentrations, the frequency of collisions between O2(X3Σ^sub g^^sup -^) and the T^sub 1^, state will be less. As such, both the rate and efficiency of O2(a^sup 1^Δ^sub g^) production will be less at these lower oxygen concentrations.

By extension, interactions between the excited state sensitizer and O2(X^sup 3^Σ^sub g^^sup -^) will also be susceptible to the magnitudes of the respective diffusion coefficients as determined by the viscosity of the surrounding medium. The effect of these parameters on O2(a^sup 1^Δ^sub g^) phosphorescence signals in photosensitized experiments has likewise been examined for sensitizers dissolved in oils and polymers (12.13).

For the present project, we set out to evaluate our single cell data in light of what is known regarding the effects of changes in O2(X^sup 3^Σ^sub g^^sup -^) concentration and solute diffusion coefficients on photosensitized O2(a^sup 1^Δ^sub g^) experiments. To this end, we also performed control experiments on sucrose solutions of different viscosity in which both the oxygen concentration and diffusion coefficient are variables. Our perspective in this project was supported by independent information indicating that intracellular oxygen diffusion coefficients and oxygen concentrations are appreciably smaller than those in neat H2O (14-16).