Label-free spatial analysis of free and enzyme-bound NAD(P)H in the presence of high concentrations of melanin.

The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.