Fluorescent TAPY–bodipy dyads as tools for imaging fungal mitochondria by confocal microscopy and flow cytometry

Mitochondria-targeted fluorescent probes are essential tools in cell biology, yet most have been optimized for mammalian cells rather than fungi, despite the growing clinical relevance of pathogenic yeasts. Here, we evaluate six triarylpyridinium-bodipy (TAPY-BDP) conjugates as mitochondrial markers in Candida albicans and Pichia kudriavzevii (formerly Candida krusei), using a triphenylphosphonium-bodipy (TPP-BDP) dyad and a non-targeted propyl-bodipy (prop-BDP) as references. Confocal microscopy and flow cytometry quantified uptake and localization. Colocalization with MitoTracker™ Deep Red gave Pearson correlation coefficients up to 0.9, supporting efficient targeting for most TAPY derivatives. TAPY-BDPs with R = H, Me, OMe, or NMe2 showed higher mitochondrial fluorescence than the more lipophilic analogues (R = Cl, CF3) and the TPP-BDP reference at 500 nM, consistent with flow cytometric analysis. Notably, TAPY(OMe)-BDP maintained robust mitochondrial staining at 100 nM in both fungi, whereas TPP-BDP decreased to near-background. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) reduced fluorescence for all cationic probes, confirming mitochondrial membrane potential-driven accumulation. Overall, these results indicate that TAPY is a competitive alternative to TPP for mitochondrial vectorization in fungi, expanding probe options for bioanalytical studies in azole-resistant yeasts and potentially enabling mitochondria-directed therapeutic delivery.