Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Abstract

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used.Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies. Video LinkThe video component of this article can be found at

On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements

“…In the case of the SLBs, external exponential correction gave D FRAP ¼ (4.0 5 0.3) mm 2 s À1 , which is larger than D FCS ¼ (2.6 5 0.2) mm 2 s À1 . D FCS is consistent with our previous results (14,23) as well as with reported values obtained by single-particle tracking (24) or equilibrating lipid gradients (25); we, therefore, consider it more reliable.…”

Preferential interactions of primary amine-terminated quantum dots with membrane domain boundaries and lipid rafts revealed with nanometer resolution

“…25,51 Mica also provides the optical transparency needed for fluorescence imaging, which was necessary for performing correlative AFM and fluorescence imaging on the same area of the bilayer. Modifying a previous approach, 41 Fig. 5 shows the successful correlation of a 60/20/20 mol% DOPC/SM/ Chol + 0.1 mol% TopFluor PC supported lipid bilayer formed on a mica substrate and imaged by both AFM and fluorescence microscopy.…”

“…To conduct correlative AFM and fluorescence imaging on supported lipid bilayers, a modified approach to a previously established method, 41 was utilized to enable fluorescence imaging on a thin layer of mica. Mica was chosen as the substrate as it enables an atomically flat surface for AFM imaging, allowing for subtle height changes due to Qdots to be detected.…”

Hydration layer of only few molecules controls lipid mobility in biomimetic membranes

“…The SLB was prepared by vesicle deposition method following formerly established protocol 49 with suitable modification. In order to form multilamellar vesicles (MLVs), 14:1 PC, SM and cholesterol in chloroform solution were mixed at a molar ratio 1:1:1 with the addition of 0.1 mol% of GM1 and 0.1 mol% of DOPE-Atto-633 to form 10 mM solution of the lipids.…”