Rapid Antifungal Susceptibility Testing by Stimulated Raman Photothermal Imaging of D ₂ O Metabolism

Invasive fungal infections pose a global health concern, fueled by the widespread use of antifungals and fungicides that have resulted in increased antifungal resistance. Timely and accurate antifungal susceptibility testing is critical not only to slow down the emergence of antifungal resistance but also to save the lives of vulnerable patients. Here, we present a proof-of-concept procedure for rapid antifungal susceptibility testing of selected <i>Candida</i> species using stimulated Raman photothermal (SRP) metabolic imaging of deuterium oxide (D₂O) at the single-cell level. The SRP platform integrates a compact, robust fiber laser with rapid and wide tuning capability and an autobalanced detection system. Owing to the high sensitivity and imaging speed of SRP microscopy, D₂O incorporation can be detected in approximately 1 h. Using this metabolic readout, susceptible and resistant strains can be determined in approximately 3 h following exposure to antifungal and D₂O assays, substantially faster than the ≥24 h required by standard broth microdilution methods, enabling rapid susceptibility assessment based on metabolic activity. Furthermore, the SRP setup and isotope labeling method are designed to be compatible with multiwell formats, suggesting potential utility as a cost-effective, high-throughput solution for future large-scale screening and research applications.