Development of a digital fluorescence sensing technique to monitor the response of macrophages to external hypoxia.

Oxygen plays a very important role in living cells. The intracellular level of oxygen is under tight control, as even a small deviation from normal oxygen level affects major cellular metabolic processes and is likely to result in cellular damage or cell death. This paper describes the use of the oxygen sensitive fluorescent dye tris (1,10-phenanthroline) ruthenium chloride [Ru(phen)(3)] as an intracellular oxygen probe. Ru(phen)(3) exhibits high photostability, a relatively high excitation coefficient at 450 nm (18 000 M(-1) cm(-1)), high emission quantum yield ( approximately 0.5), and a large Stoke shift (peak emission at 604 nm). It is effectively quenched by molecular oxygen due to its long excited state lifetime of around 1 micros. The luminescence of Ru(phen)(3) decreases with increasing oxygen concentrations and the oxygen levels are determined using the Stern-Volmer equation. In our studies, J774 Murine Macrophages are loaded with Ru(phen)(3), which passively permeates into the cells. Fluorescence spectroscopy and digital fluorescence imaging microscopy are used to observe the cells and monitor their response to changing oxygen levels. The luminescence intensity of the cells decreases when exposed to hypoxia and recovers once normal oxygen conditions are restored. The analytical properties of the probe and its application in monitoring the cellular response to hypoxia are described.