Characterization of cellular optoporation with distance
Characterization of cellular optoporation with distance
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DOI：
10.1021/ac990982u
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发表时间：
2000-03-15
影响因子：
7.4
通讯作者：
Allbritton, NL
中科院分区：
文献类型：
--
作者：
Soughayer, JS;Krasieva, T;Allbritton, NL
我们已经开发和表征细胞optoporation与可见波长的光，使用标准的未涂覆的玻璃盖玻片作为吸收介质。倍频Nd：YAG激光脉冲聚焦在玻璃表面和水性缓冲液的界面处，产生应力波并瞬时渗透附近的细胞。在贴壁细胞的光穿孔之后，存在三个空间区域，其通过细胞的活力和加载效率（或加载的细胞外分子的数量）来区分。负载效率还取决于胞外分子的浓度和分子的分子量。在离激光束最远的区域（在这些条件下>60 μ m），几乎所有的细胞都被成功加载并且存活。为了说明这种光穿孔方法的更广泛的适用性，将细胞装载蛋白激酶C的底物，然后通过毛细管电泳分析细胞内容物。与通过显微注射加载的肽相反，optopored肽显示出很少的蛋白水解降解，这表明细胞受到最小程度的扰动。也展示了未来工作的潜力，细胞被optopored并在微流体装置的封闭通道中加载荧光团。
We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (>60 mu m under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.