Autofluorescence Quenching in Decellularized Plant Scaffolds for Tissue Engineering

Purpose: Autofluorescence in plant-derived scaffolds interferes with fluorescence imaging by overlapping with commonly used fluorophores such as Hoechst and FITC. This limits the ability to visualize cell behavior and scaffold integration in tissue engineering applications. This study evaluated whether copper sulfate, ammonium chloride, or sodium borohydride can reduce autofluorescence in decellularized plant scaffolds without compromising mechanical integrity or cell viability.Methods: The effectiveness of the three quenching agents was evaluated in decellularized leatherleaf viburnum, spinach, and parsley scaffolds. Spectral scans were used to characterize baseline autofluorescence. Treated and untreated scaffolds were imaged in Hoechst, FITC, and 633 nm channels. Autofluorescence intensity, quenching stability over 24 hours, mechanical properties, and endothelial cell viability were assessed. Imaging of cell seeded scaffolds evaluated improvements in visualization after treatment.Results: Spectral scans revealed strong autofluorescence in the blue and green channels, overlapping with Hoechst and FITC. Copper sulfate reduced autofluorescence more effectively than ammonium chloride or sodium borohydride and improved nuclear visualization, with consistent performance across scaffold types. However, endothelial cell viability declined in copper-treated leatherleaf and parsley scaffolds but remained high in spinach. No significant changes in tensile strength or elastic modulus were observed after treatment.Conclusion: Copper sulfate is a highly effective and stable quenching agent for reducing autofluorescence in plant-derived scaffolds. While suitable for post-fixation imaging, scaffold-specific effects on viability limit its use in live-cell applications. Autofluorescence reduction was achieved without compromising scaffold mechanics. Ammonium chloride and sodium borohydride may be preferable when preserving cell viability is a priority.