Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization

Abstract

Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

X-ray Absorption Spectroscopy and Spectromicroscopy of Supported Lipid Bilayers

“…thesis of the lead author. 24 We also draw the reader's attention to related work on optimization of SLB formation by Ratto et al, 26 Kraft et al, 27 and Mantil et al 28 With regard to optimization of dry SLB structures, please consult the work by Seu et al 29 on surface treatments and that by Ricker et al 30 on methods to preserve phase separation through the drying process.…”

Domain Redistribution in Lipid Bilayers in the Presence of the Antimicrobial Lipopeptide Fengycin

“…SLBs are more stable than vesicles and allow for some control of lipid asymmetry within the bilayer. SLBs can be prepared using the Langmuir Blodgett technique or lipid vesicle fusion [61,65]. With the bilayer attached to a solid support (glass, mica or silicon dioxide), they can be more easily characterized with sensitive techniques.…”