Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in Candida membrane

Ergosterol-targeting amphotericin B (AmB) is the first line of defense for life-threatening fungal infections. Two models have been proposed to illustrate AmB assembly in the cell membrane; one is the classical ion channel model in which AmB vertically forms transmembrane tunnel and the other is a recently proposed sterol sponge model where AmB is laterally adsorbed onto the membrane surface. To address this controversy, we use polarization-sensitive stimulated Raman scattering from fingerprint C═C stretching vibration to visualize AmB, ergosterol, and lipid in single fungal cells. Intracellular lipid droplet accumulation in response to AmB treatment is found. AmB is located in membrane and intracellular droplets. In the 16 strains studied, AmB residing inside cell membrane was highly ordered, and its orientation is primarily parallel to phospholipid acyl chains, supporting the ion channel model. Label-free imaging of AmB and chemical contents offers an analytical platform for developing low-toxicity, resistance-refractory antifungal agents.