Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

Candida albicans, a commensal fungal pathogen, is a major cause of opportunistic infections in immunocompromised individuals. Understanding its cellular structures and pathogenic mechanisms is critical for developing targeted antifungal therapies. Stimulated emission depletion (STED) microscopy enables nanoscale visualization of cellular components, surpassing the diffraction limit of conventional light microscopy. In this study, we employed STED microscopy to investigate the ultrastructural organization of C. albicans in live specimens. We showed that dyes commonly used in STED microscopy of mammalian cells are ineffective for the study of C. albicans, and we showed the utility of Nile Red staining for visualising the organisation of dynamic cellular components, including tracking of lipid droplets, using time-lapse recording in experiments exceeding 12 hours. STED microscopy offered more than a two-fold improvement in resolution compared to confocal laser scanning microscopy applied to the same specimens with negligible photobleaching. This study demonstrates the utility of STED microscopy in advancing our understanding of C. albicans biology at the nanoscale, providing a platform for future investigations into fungal pathogenicity and antifungal development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/625149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d46837org.highwire.dtl.DTLVardef@105f661org.highwire.dtl.DTLVardef@728d11org.highwire.dtl.DTLVardef@8b65ae_HPS_FORMAT_FIGEXP M_FIG C_FIG We present an optimised fluorescence staining method for super-resolution live-cell imaging of Candida albicans, using Stimulated Emission Depletion (STED) microscopy to resolve and track sub-cellular structures. We compare the performance of conventional confocal laser scanning microscopy (CLSM) to STED imaging, providing a three-fold resolution improvement beyond the diffraction limit. Finally, we perform live cell tracking to visualise and quantify the trajectories of multiple sub-diffraction limit-sized objects over a period of 12 hours, demonstrating the potential for live-cell STED imaging of Candida to visualise key processes involved in pathogenesis, drug resistance and infection.