Abstract
Introduction: Copper ions are trace elements which act as cofactors of superoxide dismutase with antioxidant effect and is related to reduce molecular oxygen in living system. Significant alterations in Cu2+ levels in tissues have been reported in patients with various forms of malignancies. It is crucial to track Cu2+ within in a living system to explain its role in physiology. But, there was no multiphoton (MP) probe which can measure quantified value of copper ions in intact tissue without grinding or drying off. The study aim was to application of newly developed multiphoton ratiometry probe for Cu2+ with an internal reference as a tool to assess antioxidative status of colon neoplasm. Methods: We have developed a multi-photon (MP) probe for Cu2+ with an internal reference. We have employed an extended coumarin as the fluorophore (FL, red emission) having 2-picolymethylamide moiety at the 2-position as the Cu2+ chelator. FL was connected through a piperazine linker to a chromene derivative, which was employed as an internal reference (IR, blue emission). We had validated the multi-photon excited fluorescence (MPEF) intensity ratio of FL and IR so that a quantitative measurement of Cu2+ in live tissue was possible. We performed current study with validated ratiometry probe. Tissues of normal colon mucosa, colon adenoma and colon cancer obtained during colonoscopic biopsy were stained with MP probes for Cu2+ and were observed with MP ratiometry probe. Results: We could measure a quantitative measurement of Cu2+ in fresh intact colon mucosal tissue with MP ratiometry probe. The concentration of Cu2+ of colonic adenocarcinoma was 21.8uM (range 19-22.6), adenoma 13.5 uM (range12.5-14.5), normal colon mucosa 11.5 uM (range 11-12.1), respectively. Conclusion: Cu2+ quantification was possible with MP ratiometry probe in fresh intact colon neoplasm tissues. It is new method that can give help to understand cancer physiology or oxidative status in live tissues for future studies. Further study is needed to assess antioxidant status of neoplasm tissues using probe based multiphoton microscopic analysis. Figure: P-224
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