Measuring Distances in Supported Bilayers by Fluorescence Interference-Contrast Microscopy: Polymer Supports and SNARE Proteins

Abstract

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.

The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy

“…This method, termed the ''ABCD'' method, relies on thermally oxidized Si wafers into which nonoverlapping patches (sizes Ϸ10 ϫ 10 m) of different depths were etched. A similar method had been applied to measure the heights of biomembranes above surfaces (19,20,(25)(26)(27)(28)(29). For MTs, the technique is outlined in Fig.…”

Functionalization of polymerized 3D microstructures for biological applications

“…Therefore we assume that the origin of the enhancement is primarily the formation of standing waves similar to examples reported earlier (Kiessling et al 2003). Standing waves are formed by the interference of the incoming excitation beam and the excitation beam reflected by the lower gold NP layer (Fig.…”

Hydrogel Films on Optical Fiber Core: Properties, Challenges, and Prospects for Future Applications

“…Recent successful approaches to the development of new models of biological membranes by designing solid-supported phospholipid bilayers have demonstrated the importance of a water-filled space between the solid substrate and bilayer [121][122][123][124][125]. Up to now, only the planar architecture of the model has been considered.…”