Luminescent lanthanide complexes display unrivalled spectroscopic properties, which place them in a special category in the luminescent toolbox. Their long-lived line-like emission spectra are the cornerstones of numerous analytical applications ranging from ultrasensitive homogeneous fluoroimmunoassays to the study of molecular interactions in living cells with multiplexed microscopy. However, achieving such minor miracles is a result of years of synthetic efforts and spectroscopic studies to understand and gather all the necessary requirements for the labels to be efficient. This feature article intends to survey these criteria and to discuss some of the most important examples reported in the literature, before explaining in detail some of the applications of luminescent lanthanide labels to bioanalysis and luminescence microscopy. Finally, the emphasis will be put on some recent applications that hold great potential for future biosensing.

Tb‐doped La0.9Tb0.1F3 nanoparticles were prepared by a simple and reproducible microwave‐assisted synthetic protocol in water. The nanoparticles were characterized by XRD, TEM, dynamic light scattering and inductively coupled plasma atomic emission spectroscopy elemental analysis. Eleven ligands with varying coordination and photosensitizing abilities were designed to bind at the surface of the Tb‐doped nanoparticles. The photosensitizing behavior was monitored by electronic absorption spectroscopy and steady‐state and time‐resolved emission spectroscopy. The two most effective photosensitizing ligands were used to isolate and purify the capped nanoparticles. The composition and spectroscopic properties of these nanoparticles were measured, which revealed either 2660 and 5240 ligands per nanoparticle, molar absorptivities of 7.6×106 and 1.6×107 m−1 cm−1 and luminescence quantum yields of 0.29 and 0.13 in water, respectively. These data correspond to exceptional brightness values of 2.2×106 and 2.1×106 m−1 cm−1, respectively. The as‐prepared nanoparticles were imaged in HeLa cells by fluorescence microscopy, which showed their specific localization in lysosomes.