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

Figure 5

Fluorescence lifetime images of enzyme-bound NAD(P)H (𝜏₂ images) in PMNs interacting with aspergillus conidia (a) or with two clusters of aspergillus hyphae (c). Also inhere the fluorescence lifetime images were obtained by overlapping intensity images in gray scale with the spatially resolved fluorescence lifetime information in colour scale. Hence both free and phagocytosed conidia in (a) appear in gray tones indicating the absence of enzyme-bound NAD(P)H, whereas PMNs appear in colour due to the fluorescence lifetime of the enzyme-bound NAD(P)H. The increase of the fluorescence lifetime of bound NAD(P)H at the membrane of the phagosome in a PMN (enlarged detail in (a)) can be easily detected in this way. Also at the contact regions between PMNs and aspergillus hyphae (enlarged detail in (c)), the fluorescence lifetime of enzyme-bound NAD(P)H is increased. In order to quantify the increase in fluorescence lifetime of bound NAD(P)H in PMNs interacting with A. fumigatus, we performed statistics on the 𝜏₂-distributions of 90 contact regions between PMNs and hyphae and of 80 phagosome membrane regions. The results are depicted in (b) for PMNs interacting with conidia and in (d) for PMNs interacting with hyphae. Note that the average values of both distributions of increased 𝜏₂ amount to approximately 3600 picoseconds and are similar to the value determined in PMNs treated with PMA, confirming the fact that this fluorescence lifetime is specific for NADPH bound to NADPH oxidase. Furthermore, the well-defined sites of this specific lifetime in PMNs indicate the location of ROS production in these cells.