Here, we describe a method for tracking intracellular processing of siRNA-containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles (sNPs) in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes, and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.

中文翻译：

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通过共聚焦显微镜对 siRNA 的细胞内加工进行动力学分析

在这里，我们描述了一种使用自动显微镜控制和图像采集来跟踪含有 siRNA 的复合物的细胞内处理的方法，以最大限度地减少用户的工作和时间。该技术使用荧光共定位来监测由二氧化硅纳米粒子 (sNP) 在不同细胞内位置传递的双标记荧光 siRNA，包括早期/晚期内涵体、快/慢循环内涵体、溶酶体和内质网。将 siRNA 与每个细胞内位置的时间关联相结合，我们重建了 siRNA 加工中使用的细胞内通路，并展示了这些通路如何根据递送载体的化学成分而变化。