Research on synthetic delivery vectors is of major interest for cell imaging and manipulation, as they allow an efficient transfer of nucleic acids, therapeutic proteins or small drugs into the cells. We have developed a library of L-lysine analogues that allow for highly efficient gene delivery with low cytotoxicity. However little is known on the exact mechanism of uptake and the final intracellular destination of the synthetic carriers. Therefore we have developed a novel optical technique based on a modulated excitation allowing for intracellular imaging of the triplet-lifetime and -yield of fluorophores attached to the delivery vector. Both these parameters are highly dependant on the intracellular environment thus provide insight into the subcellular localization of the labelled carrier. The method combines high temporal and spatial resolution and is compatible with a multiplicity of fluorophores. We performed series of model experiments to compare the triplet lifetime and triplet yield behaviour during the natural uptake mechanism to a series of controlled conditions. The latter include microinjection of fluorescently labelled carriers directly into the cytoplasm and cell nucleus as well as in vitro measurements under conditions mimicking physiological, acidic, or DNA rich environments. To validate our technique the results from the triplet imaging were compared with two complementary methods: carrier localization by subcellular fractionation and confocal laser scanning microscopy. --- Reference --- Geissbuehler et al. Triplet imaging of Oxygen consumption during the contraction of a single smooth muscle cell (A7r5). Biophysical Journal (2010) vol. 98 (2) pp. 339-349