Revealing the ultrastructure of live Candida albicans using stimulated emission depletion microscopy.

Candida albicans, a commensal fungal pathogen, is a leading cause of opportunistic infections in immunocompromised individuals. High-resolution imaging of its cellular architecture is essential for uncovering pathogenic mechanisms and identifying antifungal targets. Here, we applied live-cell stimulated emission depletion (STED) microscopy to visualize subcellular structures in C. albicans with nanoscale precision. We found that fluorophores commonly used for STED imaging in mammalian cells were ineffective in C. albicans. In contrast, Nile Red enabled robust labelling of dynamic lipid-rich structures, including lipid droplets and intracellular membranes, with sufficient signal for long-term imaging. Using Nile Red with STED microscopy, we achieved over threefold improvement in lateral resolution (approx. 85 nm) compared to confocal microscopy, with minimal photobleaching even during continuous time-lapse acquisition over 8 h. Notably, dynamic tracking of lipid droplets revealed heterogeneous mobility patterns across individual cells, suggesting functional compartmentalization. Our findings establish STED microscopy with Nile Red staining as a powerful approach for visualizing dynamic ultrastructure in live fungal cells, expanding the toolkit for high-resolution fungal cell biology and offering new avenues for studying fungal pathogenesis and antifungal action at the nanoscale.