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.2003 Jan;84(1):408-18.
doi: 10.1016/S0006-3495(03)74861-9.

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins

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Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins

Volker Kiessling et al. Biophys J.2003 Jan.

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.

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Figures

FIGURE 1
FIGURE 1
Diagram of the tethered polymer-supported membrane systems used in this work. Oxidized silicon chips are used as substrates for polymer-supported lipid bilayers. The polymer (3400 molecular weight polyethylene glycol) is covalently attached at its two ends to the silicon oxide surface and to 3 mol % of the membrane lipids, respectively, and serves as a soft cushion between the membrane and chip surface. (A) The membrane is labeled with fluorescent lipid analogs (diI for FLIC measurements, NBD-eggPE for FRAP measurements). (B) t-SNARE receptors are reconstituted into tethered polymer-supported bilayers. After reconstitution, soluble GFP-VAMP proteins are specifically bound to the t-SNAREs in the membrane.
FIGURE 2
FIGURE 2
Fluorescence interference contrast (FLIC) microscopy. (A) Principle of fluorescence interference contrast microscopy. A fluorescent labeled membrane is supported on a patterned silicon chip with microscopic steps of silicon dioxide. The fluorescence intensity depends on the position of the dye with respect to the standing modes of the exciting and emitting light in front of the reflecting silicon surface. The position is determined by the variable height of the oxide steps and the constant width of the cleft between silicon oxide and lipid membrane. (B) Optical layer model used for the evaluation of FLIC experiments with polymer-supported membranes. Five layers are considered for the case of the fluorescent membrane: bulk silicon (refractive indexnsi, attenuation index κsi), silicon dioxide (thicknessdox, refractive indexnox), cleft (thicknessdcleft, refractive indexnH2O), stained membrane (thicknessdmem, refractive indexnmem), and bulk water (refractive indexnH2O). An additional layer is considered for the case of the fluorescent protein; namely the protein layer above the membrane surface (distance of fluorophore to membrane surfacedprot, refractive indexnH2O).
FIGURE 3
FIGURE 3
Theoretical FLIC curves as a function of oxide thickness demonstrating the effect of (A) increasing the cleft distance between the silicon dioxide and the membrane, and (B) the distance of the protein fluorophore from the membrane surface.
FIGURE 4
FIGURE 4
Supported POPC bilayer on a 16-oxide chip. (A) Fluorescence image of a 150μm × 150μm area of a polymer-supported membrane on a chip with 16 different oxide steps. Each square has a width of 2.5μm. The bilayer contains 0.5 mol % diI and was prepared by direct vesicle fusion. (B) Fluorescence intensity versus oxide thickness and the best-fit FLIC curve of the 16 oxide levels are shown in the inset. The best-fit distance isdcleft = 1.9 ± 1.0 nm and theN.A.ex is 0.86 ± 0.05. (C) Cleft distances between silicon dioxide and the membrane obtained from 40 different areas on one chip usingN.A.ex = 0.86 as a fixed parameter. The average cleft distance is 1.9 ± 0.8 nm.
FIGURE 5
FIGURE 5
Supported POPC bilayer on a four-oxide chip. (A) Fluorescence image of a 150μm × 150μm area of a supported membrane on a chip with four different oxide steps. Each square has a width of 5μm. The bilayer contains 0.5 mol % diI and was prepared by direct vesicle fusion. (B) Fluorescence intensity versus oxide thickness and the best-fit FLIC curve of the four oxide levels are shown in the inset. The best-fit distance isdcleft = 1.2 ± 1.5 nm. (C) Cleft distances between silicon dioxide and the membrane obtained from 44 different oxide quartets on one chip. The average cleft distance is 1.7 ± 1.0 nm.
FIGURE 6
FIGURE 6
Tethered polymer-supported bilayer on a four-oxide chip. (A) Fluorescence image of a 150μm × 150μm area of a tethered polymer-supported membrane on a chip with four different oxide steps. Each square has a width of 5μm. (B) Fluorescence intensity versus oxide thickness and the best-fit FLIC curve of the four oxide levels are shown in the inset. The best-fit distance isdcleft = 3.3 ± 2.0 nm. (C) Cleft distances between silicon dioxide and the membrane obtained from 45 different oxide quartets on one chip. The average cleft distance is 3.9 ± 0.9 nm.
FIGURE 7
FIGURE 7
GFP-VAMP bound to t-SNAREs in a polymer-supported bilayer on a four-oxide chip. (A) Fluorescence image of a 150μm × 150μm area of a tethered polymer-supported membrane with reconstituted t-SNAREs and bound soluble GFP-VAMP proteins on a chip with four different oxide steps. Each square has a width of 5μm. (B) Fluorescence intensity versus oxide thickness and the best-fit FLIC curve of the four oxide levels are shown in the inset. The best-fit distance between protein and the membrane surface isdprot = 16.4 ± 2.9 nm. (C) Distances between GFP and the membrane surface obtained from 59 different oxide quartets on one chip. The average distance is 16.5 ± 2.8 nm.
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References

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