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The cellular uptake mechanism and intracellular distribution of emissive lanthanide helicates have been elucidated by time-resolved luminescence microscopy (TRLM). The helicates are non-cytotoxic and taken up by normal (HaCat) and cancer (HeLa, MCF-7) cells by endocytosis and show a late endosomal–lysosomal cellular distribution. The lysosomes predominantly localize around the nucleus and co-localize with the endoplasmatic reticulum. The egress is slow and limited, around 30% after 24 h. The first bright luminescent images can be observed with an external concentration gradient of 5 mMof the EuIII helicate [Q = 0.21, t = 2.43 ms], compared to >10 mM when using conventional luminescence microscopy. Furthermore, multiplex labeling could be achieved with the TbIII [Q = 0.11, t = 0.65 ms], and SmIII [Q = 0.0038, t = 0.030 ms] analogues.