Fluorescence lifetime imaging of oxygen in living cells |
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Authors: | H C Gerritsen R Sanders A Draaijer C Ince Y K Levine |
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Institution: | (1) Department of Molecular Biophysics, Utrecht University, P.O. Box 80.000, 3508 TA Utrecht, The Netherlands;(2) TNO, Institute of Environmental Sciences, Group Sensors, P.O. Box 6011, 2600 JA Delft, The Netherlands |
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Abstract: | The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging
of oxygen in single cells was investigated utilizing fluorescence lifetime imaging. The results indicate that the fluorescence
behavior of RTDP in the presence of oxygen can be described by the Stem-Volmer equation. This shows that fluorescence quenching
by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive
to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to
quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform
fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned
throughout the cells, while the oxygen concentration is constant. |
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Keywords: | Fluorescence lifetime imaging oxygen imaging confocal microscopy macrophages |
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