The potential of using lanthanide luminescence in bioimaging has been investigated over the past decades due to the unique optical properties of the 4f-elements. By exploiting the narrow emission lines and long luminescence lifetimes, background free images with full certainty of probe localization become possible. The use of lanthanide-based bioimaging is limited by the low brightness of the Ln(III) ions and the requirement for encapsulation of the toxic ions. Use of the antenna principle combined with kinetically inert complexes for strong binding of Ln(III) ions has been a general approach to overcome the limitations of the Ln(III) ions. First, a fundamental understanding of solution structures, design guidelines, and energy migration needs to be developed to fully unravel the potential of Ln(III) ions in bioimaging. In the first part of this thesis, a detailed investigation of the solution structures of simple macrocyclic Ln(III) complexes was done. Solution properties depend on fluctuations between different conformations. To design new, efficient luminescent probes, control and insight into these fluctuations must first be understood. The rapid fluctuations in simple macrocyclic Ln(III)- complexes based on DOTA and DO3A-like ligand scaffolds were investigated by paramagnetic 1H-NMR and luminescence spectroscopy. In the second part of the thesis, three ligands for lanthanide-based probes were synthesized and characterized with bioimaging applications in mind. The challenges of fluctuations between different solution structures were highlighted in these systems, together with additional processes that complicate the energy migration. In the third and final part, a detailed photophysical study was done in different solvents and media to investigate the strong perturbations caused by the surrounding media. The three Eu(III) complexes were tested in cells on both dedicated and commercial setups. It was proven that the lanthanide complexes enter the cells and luminescence was observed in biological samples. Even so, no lanthanide luminescence was recovered on the commercial microscopes. Thus, it was concluded that lanthanide-based bioimaging still requires dedicated hardware and that other uses of Ln(III) luminescence are more readily realized.