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 ox ygen level affects major cellular metabolic processes and is likely to resu lt in cellular damage or cell death. This paper describes the use of the ox ygen sensitive fluorescent dye tris (1,10-phenanthroline) ruthenium chlorid e [Ru(phen)(3)] as an intracellular oxygen probe. Ru(phen), exhibits high p hotostability a relatively high excitation coefficient at 450 nm (18000 M-1 cm high emission quantum yield (similar to0.5), and a large Stoke shift (p eak emission at 604 nm). It is effectively quenched by molecular oxygen due to its long excited state lifetime of around 1 mus. The luminescence of Ru (phen), decreases with increasing oxygen concentrations and the oxygen leve ls are determined using the Stern-Volmer equation. In our studies, 1774 Mur ine Macrophages are loaded with Ru(phen)(3), which passively permeates into the cells. Fluorescence spectroscopy and digital fluorescence imaging micr oscopy are used to observe the cells and monitor their response to changing oxygen levels. The luminescence intensity of the cells decreases when expo sed to hypoxia and recovers once normal oxygen conditions are restored. The analytical properties of the probe and its application in monitoring the c ellular response to hypoxia are described. (C) 2001 society of Photo-Optica l Instrumentation Engineers.