Résumé
Free radicals are an important part of reactive oxygen species (ROS). They are involved in numerous cellular processes; they can act as signal transducers and their presence is essential for correct cell function. However ROS can also cause oxidative stress and they can induce cell death. Several diseases are caused by aberrant ROS production. Direct quantification of ROS remains difficult. Since their detection on cellular level is of great interest for biomedical research new detection methods are continually being developed, such as spin trapping techniques or new fluorescent probes. In this chapter we describe the potential of a new method based on the fluorescence lifetime measurement of a well-known oxygen probe: 1-pyrenebutyric acid (PBA). We record the fluorescence lifetime decays using time-resolved microfluorimetry in cells loaded with PBA. Emission is obtained through an adapted band-pass filter (404 nm) after excitation of individual cells with a pulsed nitrogen laser (337 nm). Both PBA and NAD(P)H fluorescence lifetimes are simultaneously recorded. The complex decay is resolved into three exponentials. The two short decays correspond to the free and bound NAD(P)H; the long decay corresponds to PBA. From the long decay, ROS concentrations are calculated by the Stern-Volmer equation. The NAD(P)H quantity provides information about the cell activity. We show that the addition of a second probe, Rhodamine 123, gives information on the mitochondria activity. The method is used on cells treated with sodium azide NaN(3), known to increase intracellular ROS concentration. We calculated the basal ROS concentrations for several cell types.