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We presented a low-cost and simple method to synthesize carbon nanodots (CDs) from waste wine cork using hydrothermal synthesis.  The structural and optical properties of the CDs are characterized by TEM, FTIR, Raman, UV-Vis absorption, and photoluminescence (PL) spectra. The analysis results indicated the average diameter of CDs ⁓ 6.2 ± 2.7 nm. Optical measurements showed the phenomenon of excitation-dependent PL and the formation of functional groups on the surface of the particles. CDs with a quantum yield of 1.54% was calculated using quinine sulfate as reference. Furthermore, a probe of wine cork-derived CDs in bioimaging has been successfully applied in living mesenchymal stem cells (MSCs). After treatment with CDs, MSCs exhibited fluorescence including green, yellow, and red colors under the excitation wavelengths in the range 330–385 nm, 450–480 nm, and 510–550 nm, respectively. The achievement demonstrated potential applications of fluorescent CDs in the field of the fluorescent image.

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