Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: Polymer supports and SNARE proteins
Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: Polymer supports and SNARE proteins
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DOI：
10.1016/s0006-3495(03)74861-9
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发表时间：
2003-01-01
影响因子：
3.4
通讯作者：
Tamm, LK
中科院分区：
文献类型：
--
作者：
Kiessling, V;Tamm, LK
荧光干涉对比 (FLIC) 显微镜是一种强大的新技术，用于测量距反射表面的垂直距离。氧化硅芯片表面的入射光和反射光的相长干涉和相消干涉产生不同强度的图案。通过制造具有不同高度的氧化层平台的硅芯片来探测该图案的不同水平。然后使用从沉积在这些平台上的膜收集的荧光来测量荧光探针与氧化硅表面的距离。在这里，我们应用该方法来测量支撑的脂质双层与氧化硅芯片表面之间的距离。对于普通流体磷脂酰胆碱双层，该距离为 1.7 +/- 1.0 nm。在由 3400-Da 聚乙二醇垫支撑的双层中，裂隙距离增加至 3.9 +/- 0.9 nm。该距离接近该聚合物的接枝无规卷曲所预期的弗洛里距离（4.8 nm）。在第二个应用中，测量了膜结合蛋白距膜表面的距离。整合膜蛋白 Syntaxin1A/SNAP25 (t-SNARE) 被重构为束缚聚合物支持的双层。可溶形式的绿色荧光蛋白/囊泡相关膜蛋白 (GFP-VAMP) 与重建的 t-SNARE 结合。 GFP 距膜表面的距离为 16.5 +/- 2.8 nm，表明棒状 t-SNARE/v-SNARE 复合物与膜表面的垂直方向。
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.