The purines ATP, ADP, and adenosine are important extracellular signaling agents. Analysis of purinergic signaling has been slowed by lack of direct methods for measurement of purine release in real-time during physiological activity. We have previously reported microelectrode biosensors for adenosine, but similar sensors for ATP have remained elusive. We now describe an ATP biosensor formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3-phosphate oxidase. It responds rapidly (10-90% rise time <10 s) and exhibits a linear response to ATP over the physiologically relevant concentrations of 200 nM-50 microM and is very sensitive approximately 250 mA.M(-1).cm(-2). By including phosphocreatine kinase in the biolayer, we can optionally amplify the ATP signal and also make the sensor sensitive to external ADP. We have used our sensors to make the first demonstration that ATP is released from spinal networks in vivo during locomotor activity.

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.

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Time-resolved singlet oxygen, O(2)(a(1)Delta(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 H(2)O. This conclusion is supported by O(2)(a(1)Delta(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 O(2)(a(1)Delta(g)) is comparatively long ( approximately 3 micros) and does not differ significantly from that in neat H(2)O ( approximately 3.5 micros). Despite this long lifetime, however, our estimate of an apparent intracellular oxygen diffusion coefficient in the range approximately 2-4 x 10(-6) cm(2) s(-1) means that the spatial domain of intracellular O(2)(a(1)Delta(g)) activity will likely have a spherical radius of approximately 100 nm. This latter point helps reconcile seeming inconsistencies between our direct O(2)(a(1)Delta(g)) lifetime data and results obtained from independent photobleaching experiments that show a limited translational diffusion distance for O(2)(a(1)Delta(g)) within a cell.

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. Time- and spatially-resolved optical measurements of both singlet oxygen and its precursor, the excited state sensitizer, reflect the complex and dynamic morphology of the cell. These experiments help elucidate photoinduced, oxygen-dependent events that compromise cell function and ultimately lead to cell death. In this perspective, recent work on the photosensitized production and detection of singlet oxygen in single cells is summarized, highlighting the advantages and current limitations of this unique experimental approach to study an old problem.