A CandiChrome toolkit for multicolor labeling of Candida cells

Here, we develop CandiChrome, a multiplex labeling toolkit for Candida albicans , through the 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. 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 analysis identified a CandiChrome panel with two practical implementations: a seven-fluorophore panel for microscopy-based imaging and a six-fluorophore panel for flow cytometry-based applications. Moreover, this platform could resolve up to 21 distinct populations by flow cytometry and microscopy when used with spectral demixing. 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 the CandiChrome panel as a flexible platform for multiplex fungal imaging and flow cytometric analysis in a pathogenic species where multicolor tools remain underdeveloped.