Transient Absorption Microscopy, Instrumentation and Application to Amphotericin B

Transient absorption microscopy (TAM) has emerged as a highly useful tool for studying a wide range of applications ranging from materials to biology. It has the advantage of being able to stimulate signal from non-fluorescent molecules and through microscopy is able to image these molecules without reliance on fluorescent quantum yield. A transient absorption microscope has been fully constructed and an initial instrumentation study is presented with white light compression and imaging of polystyrene beads in a solution of IR 1-44 laser dye. We are utilizing TAM for the study of the small-molecule antifungal, Amphotericin B (AmB) and how it interacts with living cells. There have been multiple studies conducted which present conflicting reports of the mechanism of action in which AmB is killing fungus. By directly imaging the AmB molecules interacting with the cells, we will be able to more clearly determine which, if any, of the previously proposed mechanisms is accurate. We have been able to image what we believe is AmB being internalized into yeast cells which is contradictory to the previously proposed mechanisms. While we have obtained preliminary images of the drug interacting with living cells, further investigations are necessary to obtain a full picture of the interactions with AmB and fungal cells.