Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging
Figure 2
(a)
3D and 2D images obtained by means of steady-state TPLSM showing the endogenous
fluorescence of PMNs. The endogenous fluorescence intensity is low in the
nuclei and high in small organelles around the nucleus supposed to be
mitochondria. (b) Endogenous fluorescence (green, left) and Rhodamine 123
fluorescence (red, centre) are shown to be colocalised in living PMNs (yellow,
right). Since Rhodamine 123 accumulates in the mitochondria of living cells, we
can conclude that the bright structures in the autofluorescence image are
mithocondria. (c) Fluorescence decay curves in different PMNs 10 minutes after
NaCN addition (30 mol/L). While in some cells (left diagram) the decay is
monoexponential, that is, only free NAD(P)H, in other cells (right diagram), a
biexponential behaviour of the decay has been retrieved, that is, both free and
enzyme-bound NAD(P)H is monitored. These results indicate that under the given
experimental conditions ( and fluorescence detection at 460 nm), the endogenous cellular fluorescence mainly originates from the coenzymes
NADH and NADPH.
