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To improve visualization of nanoparticles within the cells’ compartments, we synthesized a coumarin based fluorescent derivative, tetradecyl diethylamino coumarin amid, 14-DACA. In this compound the coumarin chromophore is linked with a tetradecyl alkyl chain that contributes to lipophilicity and slightly amphiphilic character of this probe. 14-DACA exhibits good biocompatibility, its solubility and emission spectrum are not sensitive to changes in pH value. Solid lipid nanoparticles (SLN) labeled with 14-DACA (SLN-D) and frequently used 6-coumarin (SLN-C) were utilized to evaluate probes’ properties in the trafficking and intracellular localization of nanoparticles. SLN-D were seen as distinct blue dots in the cellular environment in contrast to SLN-C which were hardly to recognize due to the self-quenching of 6-coumarin, its leakage and distribution in intracellular compartments. Spectra of 14-DACA indicated the possibility of spectral resolution from both green and red fluorophores allowing clear multicolor imaging of organelles in both fixed and living cells. The results showed valuableness of new probe for trafficking of the drug nanocarriers intracellularly in a kinetic and sensitive manner. Such studies are of great importance in investigations aimed to clarify subcellular targeted drug delivery, controlled release and even to identify toxicological changes.