New research sheds light on Candida albicans yeast – the most common cause of fungal infections worldwide – aiming to improve efforts in combatting antimicrobial resistance (AMR). To learn more about this initiative, we connected with lead researcher Gail McConnell, Professor at Strathclyde Institute of Pharmacy and Biomedical Sciences.
Why was Candida albicans chosen as the model organism for this study?
Candida albicans is both a commensal organism and a major cause of opportunistic fungal infections, particularly in immunocompromised patients. Despite its clinical importance, many aspects of its subcellular architecture have remained poorly understood because existing imaging techniques cannot resolve dynamic structures in living cells. By applying live-cell STED microscopy, we sought to bridge the gap between static, high-resolution electron microscopy (EM) – useful for detailed views of fixed specimens – and lower-resolution fluorescence imaging, which enables live-cell observation but lacks fine spatial detail. Our work reveals how key organelles, including lipid droplets and mitochondria, behave in the living pathogen, providing insight into cellular processes that contribute to pathogenicity and antifungal resistance.
What advantages does STED microscopy offer over traditional EM or confocal methods for studying fungal pathogens?
EM provides exceptional structural detail but requires fixation, dehydration, and vacuum conditions. Because these steps kill cells, the technique captures only a single static snapshot, offering no information about how structures move or change over time. It also cannot distinguish between molecular species without additional multimodal approaches. Confocal microscopy, by contrast, offers molecular specificity and is well suited for live-cell imaging, but its resolution is limited by light diffraction to around 200 nm. As a result, smaller structures cannot be resolved clearly, making their behavior difficult to study.
STED microscopy addresses these limitations by combining high spatial resolution with live-cell compatibility. In our study, it achieved nanoscale resolution of approximately 100 nm while maintaining cell viability for several hours. This allowed us to visualize and track organelles and membrane interactions dynamically, providing both spatial and temporal precision – capabilities essential for understanding fungal behavior under physiological conditions or during antifungal treatment.
What were the main technical challenges you faced when adapting STED microscopy for live fungal imaging?
STED microscopy has been highly effective for studying mammalian cells, but in our work the thick fungal cell wall posed a significant challenge. Unlike the relatively thin plasma membrane of mammalian cells, the Candida albicans cell wall is a much more substantial barrier, preventing many standard STED fluorophores from entering the cell. As a result, commercial dyes designed for live-cell STED imaging in mammalian systems produced little to no usable signal in Candida albicans.
We addressed this limitation by identifying Nile Red, a small, neutral, lipophilic dye capable of crossing the fungal cell wall and labeling intracellular lipid-rich structures. By optimizing dye concentration, washing steps, and imaging parameters, we achieved stable, long-term labeling with minimal phototoxicity or photobleaching during eight hours of continuous imaging. Because Nile Red is widely used in conventional light microscopy and commonly available in laboratories, it offers an accessible solution for other researchers seeking to apply live-cell STED methods to fungal studies.
What were the main outcomes of the study – did anything surprise you?
We achieved more than a threefold improvement in resolution compared with confocal microscopy, allowing us to visualzse organelle ultrastructure and dynamics that had not previously been observed in live Candida albicans. One of the most unexpected findings was the heterogeneous mobility of lipid droplets within individual cells. This pattern suggests a level of subcellular organization not previously documented in this pathogen and highlights how Candida albicans may dynamically manage its internal architecture, potentially as a survival strategy.
How might this approach help clinical laboratories better understand antifungal resistance mechanisms or the cellular effects of drug treatments?
By enabling nanoscale visualization in live fungal cells, this technique opens new possibilities for tracking how antifungal agents affect membrane organization, vacuole function, or lipid metabolism in real time. Lipid droplets, for example, are known to sequester toxins and contribute to drug tolerance. With STED microscopy, clinicians and researchers can observe these processes directly, helping to identify cellular responses to drug exposure, mechanisms of resistance, or even early biomarkers of treatment efficacy.
How accessible is STED microscopy for diagnostic or research laboratories, in terms of cost, training, and required expertise?
STED systems remain relatively specialized instruments that require significant investment and technical expertise. However, newer turnkey platforms, such as the system used in our study, are far more compact and user-friendly than earlier generations. Training typically involves understanding fluorophore compatibility, photophysics, and image processing – areas that are increasingly supported by technical experts in core microscopy facilities.
In your view, what role can advanced microscopy play in bridging the gap between basic fungal biology and clinical diagnostics?
The opportunities are substantial. Advanced imaging provides the spatial context needed to connect molecular findings with functional cell biology. In fungal diagnostics – where morphology and biofilm structure can influence virulence and treatment outcomes – nanoscale imaging offers a way to correlate structural states with clinical behavior. By visualizing live-cell responses to stress, immune factors, or drugs, super-resolution microscopy can help bridge molecular microbiology with improved diagnostic approaches and more informed therapeutic strategies.
How do you envision pathologists and microbiology laboratories using nanoscale imaging data to inform antifungal therapy or infection control strategies in the future?
Pathologists may soon be able to assess how antifungal agents affect cell wall integrity or vacuolar dynamics at the nanoscale, providing an additional dimension to antifungal susceptibility testing. Imaging could reveal, for example, whether a strain forms resistant biofilm structures or alters lipid trafficking pathways under drug pressure. Such visual “fingerprints” could complement molecular resistance assays, support more personalized antifungal therapy, and help guide infection control strategies in healthcare settings.
What are your next steps in refining this methodology?
Our next steps involve integrating environment-sensitive fluorescent probes, described by Danylchuk, Jouard, and Klymchenko, to visualize changes in lipid order and membrane dynamics in live Candida albicans under stress or antifungal treatment. By combining these probes with multicolor and STED imaging, we aim to track multiple organelles simultaneously and monitor their biophysical states in real time. This will extend our approach from purely structural imaging to functional nanoscale analysis, providing new insights into how fungal cells remodel their membranes during drug exposure and infection.
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