To demonstrate the applicability of the long fluorescence lifetime of Nuc-DAOTA + for imaging studies, the fixed 3T3 cells co-stained with Nuc-DAOTA + (Ex/Em = 560/600 nm) and Mito-Tracker Red CM-H2XRos (Ex/Em = 579/599 nm) and the FLIM images were captured on a home-built setup. Upon excitation at 520 nm and capturing emission above 561 nm, we mainly observed fluorescence from the nuclei of the fixed cells (Figure ). The corresponding FLIM image clearly showed that the nuclei are associated with the longer fluorescence lifetime of Nuc-DAOTA + , while emission from Mito-Tracker Red give rise to a shorter fluorescence lifetime evenly spread throughout the cells (Figure ). While the FLIM image depicts the intensity averaged fluorescence lifetime (calculated from biexponential fits), which are shorter than the expected ~ 20 ns (from solution measurements), the individual fitting components reveal that the longer lived component takes values as high as 16 ns (Figure ). Additional imaging examples even shown that the long-lived fitting component reached values of 20 ns (Figure ). Masking the images to only consider the regions of the nuclei displayed similar fluorescence lifetime distributions as for the entire image (Figure ).