The main theme of this thesis is to develop a fluorescent probe for imaging the subcellular distribution of kinetically labile copper pools that might play a critical role in copper homeostasis. Various copper-selective sensors were designed by combining 1,3,5-triaryl-2-pyrazoline fluorophores with polythioethers as receptor moieties. A series of donor-substituted 1,3,5-triaryl-2-pyrazoline fluorophores were synthesized and characterized in terms of their photophysical and electrochemical properties. Interestingly, the aryl substituents attached to the 1- and 3-position of the pyrazoline ring influence the photophysical properties of the fluorophore in distinctly different ways. The excited-state equilibrium energy is primarily influenced by changes of the substituent in the 1-position, whereas the reduction potential of the fluorophore is determined by the 3-aryl group. Results from computational analyses agree well with the experimental data. A pyrazoline fluorophore library was synthesized, and their photophysical and electrochemical properties were studied. The compounds cover a broad range of excited state energies and reduction potentials, and allow for selective and differential tuning of these two parameters. A series of thiazacrownethers and tripodal aniline copper(I) receptors were synthesized and their copper binding stoichiometries, stability constants, and copper-self-exchange kinetics were investigated. The measured self-exchange activation parameters revealed for all studied ligands a negative activation entropy, suggesting a predominant associative exchange mechanism. With detailed knowledge of the fluorophore platform and copper receptors, sensor CTAP-1 was designed, synthesized and characterized. The probe shows a 4.6-fold emission enhancement and reaches a quantum yield of 14% upon saturation with Cu(I). The sensor exhibits excellent selectivity towards Cu(I) and is insensitive towards millimolar concentrations of Mg(II) or Ca(II). Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular location of the mitochondria and the Golgi apparatus. The subcellular topography of copper was further determined by synchrotron-based x-ray fluorescence (SXRF) microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper. The data provide a coherent picture with evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.