Using Second Harmonic Generation to Study Gram-Positive Bacterial Membranes

Understanding how small-molecules, such as drugs, interact with bacterial membranes can quickly unravel into much more perplexing questions. No two bacterial species are alike, especially when comparing their membrane compositions which can even be altered by incorporating fatty acids from their surrounding environment into their lipid-membrane composition. To further complicate the comparison, discrete alterations in small-molecule structures can result in vastly different membrane-interaction outcomes, giving rise to the need for more "label-free" studies when analyzing drug mechanisms. The work presented in this dissertation highlights the benefits to using nonlinear spectroscopy and microscopy techniques for probing small-molecule interactions in living bacteria. A large aim of this work focuses on the theory and applications to utilizing two nonlinear optical phenomena, second harmonic generation (SHG) and two-photon fluorescence (TPF), for understanding environmental and lipid-membrane composition effects on small-molecule uptake and transport in bacterial systems, such as: Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. SHG and TPF simultaneously monitored the uptake and transport of two membrane-associated probe molecules in two gram-positive bacterial species, E. faecalis and S. aureus. Only SHG was employed for monitoring the gram-positive antibiotic, daptomycin, interactions under different environmental conditions and calcium-ratios with the gram-positive species E. faecalis and S. aureus, and the gram-negative species E. coli as a control.