Fluorescence lifetime measurements in confocal microscopy of neurons labeled with multiple fluorophores

In order to resolve multiple fluorophores by their lifetimes in discrete tissue domains, the labeling intensity must be sufficiently strong and the intensity-difference between the labels must not be too large, the rate of fading should be similar for all fluorophores, and the lifetimes of the fluorophores should be sufficiently discrete. We could readily distinguish Cyanine-3.18 (Cy-3), Lissamine Rhodamine (LRSC), and Texas Red when they were not colocalized in tissue profiles. Colocalization of Cy-3 and LRSC, as well as Cy3 and Texas Red, could also be distinguished, while the combination of LRSC and Texas Red was more difficult. We have used fluorescence lifetime recordings in confocal microscopy to detect different neuropeptides in neurons. We demonstrate that somatostatin and galanin are colocalized in axon profiles of the spinal cord dorsal horn.