Ke, C.-L.; Xu, J.; Frazer, C.; Bennett, R. J.

Here, we develop CandiChrome, a multiplex labeling toolkit for Candida albicans, through combined in vitro and in vivo characterization of fluorescent proteins in a standard strain background. To this end, we screened 13 candidate fluorophores across the visible spectrum and assessed their practical performance based on brightness, stability, and usability. This analysis identified a seven-fluorophore set that achieved the most effective balance of signal strength, robustness, and compatibility. We used this optimized panel to build a modular multicolor platform that enables strain labeling, mixed-population imaging, and competition assays in C. albicans. This platform could resolve up to 21 distinct populations by flow cytometry and microscopy. Importantly, CandiChrome supported the resolution of differentially labeled populations both in vitro and in the murine host, supporting the simultaneous tracking of multiple strains in complex settings. Together, these results establish CandiChrome as a flexible platform for multiplex fungal imaging in a pathogenic species where multicolor tools remain underdeveloped.