Domain structure in model membrane bilayers investigated by simultaneous atomic force microscopy and fluorescence imaging
Domain structure in model membrane bilayers investigated by simultaneous atomic force microscopy and fluorescence imaging
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DOI：
10.1021/la030175x
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发表时间：
2003-09-30
期刊：
影响因子：
3.9
通讯作者：
Burns, AR
中科院分区：
文献类型：
--
作者：
Burns, AR
利用原子力显微镜（AFM）和亚微米共聚焦荧光成像技术研究了1，2-二油酰-sn-甘油-3-磷酸胆碱（DOPC）中1，2-二棕榈酰-sn-甘油-3-磷酸胆碱（DPPC）的脂质结构域。在头基或尾基处由荧光探针标记的脂质基于凝胶（DPPC）和无序液体（DOPC）相之间的分配在荧光成像中实现域对比。然而，与AFM地形信息的相关性表明，它们并不总是忠实地报告准确的凝胶域的大小或形状。此外，我们发现，荧光对比度显着降低域的大小，这样，尽管有足够的光学分辨率与原子力显微镜观察到的小域没有观察到荧光图像。我们将这些影响部分归因于探针脂质在域边界上的加宽分区。荧光Alexa 488结合的霍乱毒素B亚基GM 1神经节苷脂在DPPC域的结合与AFM地形信息的光学分辨率的限制。然而，它也可以揭示在没有地形对比度的流体相中存在稀释的GM 1组分。在所有情况下，地形和荧光图像的完整相关性提供了证据，凝胶相域发生在双层的两个小叶。
Simultaneous atomic force microscope (AFM) and submicron confocal fluorescence imaging of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domain structures in 1,2-dioleoyl-sn-glycero3-phosphocholine (DOPC) is presented. Lipids labeled by fluorescent probes either at the headgroups or tailgroups enable domain contrast in fluorescence imaging on the basis of partitioning between the gel (DPPC) and disordered liquid (DOPC) phases. However, correlation with AFM topographic information reveals that they do not always faithfully report exact gel domain size or shape. Furthermore, we find that the fluorescence contrast decreases significantly with domain size, such that small domains observed with AFM are not observed in fluorescence images despite adequate optical resolution. We attribute these effects in part to broadened partitioning of the probe lipids across the domain boundaries. Binding of fluorescent Alexa 488-conjugated cholera toxin B subunits to GM1 gangliosides in DPPC domains correlates well with AFM topographic information to the limit of optical resolution. However, it also may reveal the presence of dilute GM1 components in the fluid phase that have no topographic contrast. In all cases, the complete correlation of topographic and fluorescence images provides evidence that gel-phase domains occur across both leaflets of the bilayer.