Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

Figure 4

Fluorescence lifetime images of enzyme-bound NAD(P)H (𝜏₂ images) in naive PMNs (a), in PMNs after addition of PMA (NADPH oxidase activator) (b), in PMNs after addition of AEBSF (NADPH oxidase inhibitor) (c), and in PMNs after addition of AEBSF followed by PMA as a negative control of the stimulation (d). Note that the fluorescence lifetime after addition of PMA (Figure 4(b))—especially in the membrane regions (Figure 4(h))—is longer than in the other images (Figures 4(a), 4(c), 4(d)). The increased average fluorescence lifetime after the stimulation with PMA is confirmed by the corresponding 𝜏₂-histograms in (e). Statistics over regions with increased 𝜏₂ in PMNs treated with PMA allowed a quantification of the fluorescence lifetime corresponding to the NADPH bound to NADPH oxidase (Figure 4(f)). The 𝜏₂-distribution in each region of increased fluorescence lifetime in PMNs treated with PMA was evaluated in order to identify the specific fluorescence lifetime peak of NADPH bound to NADPH oxidase as shown in (g). Figure 4(h) depicts the overlapp of 𝜏₂-maps of PMNs activated with PMA (green) and of regions of NOX2-specific fluorescence lifetime (3670±170 picoseconds) in the same cells (white). All fluorescence lifetime images were obtained by overlapping intensity images in gray scale with the spatially resolved fluorescence lifetime information in colour scale.