Label-Free Visualization of the Antifungal Polyene Drug, Nystatin, in Biological Membranes Using Raman Microscopy

Polyene macrolides are a class of antifungal drugs that remain essential for treating life-threatening fungal infections. The complex relationship between the chemical structure of the polyene macrolide, its interaction with biological membranes and subsequent intracellular localization, is poorly understood. Nystatin is an archetypal polyene macrolide, which has been in clinical use for the treatment of oral and dermal Candida albicans infections over several decades. To investigate the interaction of nystatin with biological membranes, we applied label-free stimulated Raman scattering (SRS) microscopy for this purpose. In mammalian cells, the formation of large extracellular aggregates of nystatin were observed, together with binding to the plasma membrane. In C. albicans, SRS imaging revealed a significant accumulation of nystatin in the fungal membrane and the internalization of the drug. Spectral phasor analysis was applied to identify the discrete nystatin signal from the cellular content using hyperspectral SRS imaging. This work provides the first label-free and direct visualization of nystatin binding in biological membranes and the subsequent trafficking in fungal cells using SRS microscopy.