Beyond Single-Stain Controls in Plant-Based Cheese Microstructure Analysis: A tri-phase approach for validating multiplex food microscopy

The development of plant-based cheese alternatives requires robust analytical methods for microstructural characterisation of starch-containing systems. This study established a validated confocal laser scanning microscopy protocol for simultaneous visualisation of protein, fat, and starch components through systematic evaluation of fluorescent staining methodologies. A systematic tri-phase experimental approach, progressing from single-component gels through binary to ternary systems, validated staining specificity across all fluorescent probes. Calcofluor White (CFW), established for visualising β-1,3 and β-1,4 linked polysaccharides (cellulose, chitin, β-glucans), were evaluated for binding α-linked starch polysaccharides (amylose and amylopectin) in cheese matrices. However, commercial CFW preparations containing Evans Blue demonstrated significant cross-reactivity with proteins, compromising analytical accuracy. Fluorescent Brightener 28 was identified as an effective alternative, providing selective starch labelling without protein interference. This research provides a robust methodology for simultaneous three-component visualisation in plant-based cheese systems, enabling accurate microstructural analysis essential for product development in this rapidly expanding market segment.