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Copper ions are essential for many biological processes. However, high concentrations of copper can be detrimental to the cell or organism. A novel naphthalimide derivative bearing a monoboronic acid group (BNP) was investigated as a Cu2 selective fluorescent sensor in living cells. This derivative is one of the rare examples of reversible fluorescent chemosensors for Cu2 which uses a boronic acid group for a binding site. Moreover, the adduct BNP-Cu2 displays a fluorescence enhancement with fructose. The uptake of this novel compound in HeLa cancer cells was imaged using confocal fluorescence microscopy techniques including two-photon fluorescence lifetime imaging microscopy.