Copper participates in a range of critical functions in the nervous system and human brain. Disturbances in brain copper content is strongly associated with neurological diseases. For example, changes in the level and distribution of copper are reported in neuroblastoma, Alzheimer's disease, and Lewy body disorders, such as Parkinson disease and dementia with Lewy bodies (DLB). There is a need for more sensitive techniques to measure intracellular copper levels to have a better understanding of the role of copper homeostasis in neuronal disorders. Here, we report a reaction-based near-infrared (NIR) ratiometric fluorescent probe CyCu1 for imaging Cu 2+ in biological samples. High stability and selectivity of CyCu1 enabled the probe to be deployed as a sensor in a range of systems, including SH-SY5Y cells and neuroblastoma tumors. Furthermore, it can be used in plant cells, reporting on copper added to Arabidopsis roots. We also used CyCu1 to explore Cu 2+ levels and distribution in post-mortem brain tissues from patients with DLB. We found significant decreases in Cu 2+ content in the cytoplasm, neurons, and extraneuronal space in the degenerating substantia nigra in DLB compared with healthy age-matched control tissues. These findings enhance our understanding of Cu 2+ dysregulation in Lewy body disorders. Our probe also shows promise as a photoacoustic imaging agent, with potential for applications in bimodal imaging.