A fluorescence imaging protocol for correlating intracellular free cationic copper to the total uptaken copper by...

A variety of fluorescence probes have been developed for fluorescence imaging of metals in biological cells. However, accurate quantification of metals with fluorescent approaches is challenging due to the difficulty in establishing a standard calibration curve in living cells. Herein, a fluorescence imaging protocol is developed for imaging intracellular Cu²⁺ and its correlation with the cellular uptake of copper. The total amount of intracellular Cu is detected by inductively coupled plasma mass spectrometry (ICP-MS) in parallel. Fluorescence imaging of Cu²⁺ is accomplished with Rhodamine B derivative modified carbon dots (CDs-Rbh) based on fluorescence resonance energy transfer (FRET) from CDs to rhodamine. Intracellular Cu²⁺ is correlated with fluorescence ratio at λ_em 500-600 nm (rhodamine) to λ_em 425-475 nm (CDs) with excitation at λ_ex 405 nm. It is found that Cu²⁺ is linearly correlated with the total intracellular uptaken copper content, with a linear correlation between the relative fluorescence ratio in fluorescence imaging and intracellular Cu derived from ICP-MS, including both Cu(I) and Cu(II) species. The linear calibration equation is lg(F₂/F₁) = 0.00148 m[Cu]-0.3622. This approach facilitates further investigation and elucidation of copper transition in live cells and the evaluation of their cytotoxicity.