In most cases, paramagnetic centres are introduced via site-directed spin-labelling (SDSL) of specific residues, usually employing stable organic radicals such as nitroxides or trityls, or paramagnetic metal ions such as Gd III , Mn II , or Cu II . In most cases, SDSL involves stable radicals introduced via cysteine-specific chemistry, where the label-specific length of the linker contributes degrees of freedom to the dynamics of the protein, which artificially broadens distance distributions or allows only distinct label conformations that could make interpretation of distance distributions ambiguous. Additionally, different spin labels may lead to different degrees of perturbation of native structure due to their size and interactions with protein residues, e.g. trityl labels. Another approach uses Cu II complexed with nitrilotriacetic acid (CuNTA) coordinated to a site-specifically introduced double-histidine (dHis) motif (dHis-CuNTA), posing more stringent requirements on the labelling site but yielding very narrow distance distributions due to the rigidity of this labelled side-chain. CuNTA increases specificity to dHis sites compared to free Cu II in solution, which has higher propensity to unspecific binding. Depending on the secondary structural elements (-helix or -sheet), dissociation constants (Kd) were determined to be in the order of 10 -5 to 10 -7 under EPR conditions. An advantage of using spectroscopically orthogonal labels is that binding sites can be saturated by an excess of CuNTA without overlap with the detected signal. Furthermore, the dHis-CuNTA labelling has been shown to be robust against competing ligands and retaining its high affinity binding over a wide pH range, thus demonstrating biologically relevant compatibility. While the majority of the benchmarking studies on dHis-CuNTA have been performed on different constructs of a model protein (GB1, vide infra), this labelling approach has also been applied to a variety of more complex biological systems. Despite its vulnerability to reduction CuNTA has been used for in-cell PDS. Sensitivity is of crucial importance for PDS studies. Over the last decade, the research questions investigated with PDS have involved ever larger biomolecular complexes. It is conceivable that these are concentration-limited, either due to poor solubility or low expression yield. Studying the dispersed state of proteins undergoing liquid-liquid phase separation at micromolar concentrations requires submicromolar sensitivities. Furthermore, physiologically relevant conditions normally require high-sensitivity techniques due to the generally low concentrations of most biomolecules in cellulo or in vivo. Recent benchmarking studies have demonstrated that PDS allows measurements at nanomolar spin concentration in vitro and in-cell. In one study, spectroscopically orthogonal labelling using dHis-CuNTA and the nitroxide MTSL in combination with RIDME measurements yielded reliable distance information in the short-to-medium distance range down to 100 nM protein concentration in vitro, and similar concentration limits were reported for in vitro nitroxide-nitroxide and in-cell Gd III -Gd III PELDOR measurements. Recent studies reported DQC based distance measurements at protein concentrations of 25 nM (short distance range) and 45 nM (long distance range) doubly-labelled with MTSL 1 or oxSLIM, respectively. At sufficiently low temperature and spin concentration the electron spin dephasing time becomes concentration independent, and the achievable distance range is largely limited by deuteration levels of the sample. With the commonly used large volume resonators and rectangular pulses, one limiting factor for sensitivity of nitroxides is the excitation bandwidth; here, labels with a narrow spectral line width such as trityls could entail a factor 3 to 4 in sensitivity gain by allowing to excite the full spectrum.