With the aim to quantify rhodamine-labeled plasmid DNA in cellular compartments while avoiding problems arising from subcellular fractionation, like recovery and leakage, Akita et al. [173] developed a novel quantitative strategy, the confocal image-assisted three-dimensionally integrated quantification (CIDIQ) method. To distinguish endosomes/lysosomes and the nucleus from the cytosol, they were stained with LysoSensor DND-189 and Hoechst 33258, respectively, and sequential Z-series images were captured by CLSM. By applying this quantification method, the authors could show that due to a rapid endosomal escape, Lipofectamine Plus delivered more plasmid into the nucleus than R8 or stearylated R8. Hama et al. [174] used the same method in combination with TaqMan PCR to evaluate the uptake and intracellular distribution of plasmid DNA after delivery with viral as well as non-viral vectors. Due to superior cell surface binding, the efficiency of cellular uptake was significantly higher for Lipofectamine Plus than for adenovirus whereas intracellular trafficking, i.e. endosomal escape and nuclear transfer, were essentially the same. However, to achieve comparable transgene expression, 8000-fold higher intranuclear plasmid numbers were required in case of Lipofectamine Plus. This finding suggests a difference in nuclear transcription efficiency after non-viral delivery.