SCANNING TRANSMISSION X-RAY AND FLUORESCENCE MICROSCOPY OF LIPID BILAYERS

Abstract

Cationic antimicrobial peptides (cAMPs) are of interest as a possible solution to the problem of bacterial resistance to antibiotics. In order to facilitate identification of useful cAMPs, the mechanism of action in killing prokaryotic cells is of interest. Robert Hancock (UBC) has proposed that charge interaction between cAMPs and the negatively-charged bacterial membrane is a major factor that contributes to the disruption of the bacterial membrane. The goal of this thesis is to contribute to the development of a method based on scanning transmission X-ray microscopy (STXM) to investigate the electrostatic interaction hypothesis as the method by which cAMPS interact with negatively charged bacterial membranes, using fluorescence microscopy (FM) as a guide. Methods were developed to generate phase-segregated lipid bilayers as model membranes on the silicon nitride membranes. C 1s, N 1s and O 1s X-ray absorption spectra of 3 lipid species - 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC), 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt) (DOTAP) were obtained to be used as reference standards in future studies. FM and STXM were used to map the saturated and unsaturated domains in dried lipid bilayers exhibiting phase segregation. Attempts were also made to image the lipid bilayers under hydrated conditions using both static and flow cells. Efforts to develop a flow cell for STXM using 3D printing are outlined. The potential to evolve this line of research to enable systematic studies of protein and peptide interactions with lipid bilayers under static and dynamic conditions is discussed.