Posing for a picture: vesicle immobilization in agarose gel.
Posing for a picture: vesicle immobilization in agarose gel.
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DOI：
10.1038/srep25254
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发表时间：
2016-05-03
影响因子：
4.6
通讯作者：
Riske KA
中科院分区：
文献类型：
--
作者：
Lira RB;Steinkühler J;Knorr RL;Dimova R;Riske KA
拍摄照片通常需要感兴趣的对象静止不动。在科学上，光学和电子显微镜增强了成像。然而，生命和软物质并不是静止的。因此，用于显微镜检查的生物制剂通常包括固定步骤。类似地，固定化策略是细胞或脂质囊泡成像所需的或基本上促进细胞或脂质囊泡成像，并且甚至更是通过基于荧光的技术获取高质量数据所需的。在这里，我们描述了一种简单而有效的方法，如使用琼脂糖凝胶的大小在0.1和100 μm之间的脂质囊泡。我们发现，虽然大的和巨大的单层囊泡（LUV和GUV）可以被关在凝胶网络的口袋里，小分子，蛋白质和胶束仍然可以自由地通过凝胶扩散，并与膜相互作用，如在无琼脂糖的解决方案，和复杂的生化反应，涉及几种蛋白质可以在凝胶中进行。同时，在琼脂糖中的固定化对GUV的大小和稳定性没有不利影响。通过应用技术，如FRAP和FCS，我们表明，脂质的横向扩散不受凝胶。最后，我们的固定策略允许捕获GUV的高分辨率3D图像。
Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs.