Department of Developmental and Molecular Biology and Department of Genetics, Albert Einstein College of Medicine , New York, NY, USA.
Autophagy. 2020 Sep;16(9):1711-1717. doi: 10.1080/15548627.2019.1695400. Epub 2019 Nov 27.
The endolysosomal system is critical for protein homeostasis in cells. A common way of studying protein transport and degradation (e.g. via autophagy) is by labeling vesicular structures such as endosomes, autophagosomes, lysosomes, or model substrates with fluorescent tags or by fluorescent antibody staining. Detailed analyses require quantification of hundreds of structures under various conditions. Typically, the images are analyzed individually with software such as the widely available Fiji/ImageJ (https://imagej.net/Fiji/Downloads), adjusting and thresholding each image and channel independently, which is a very labor intensive and fastidious task. To streamline the process, we developed a plug-in that, integrated into Fiji, enables the automated quantification of vesicular (i.e. punctate) structures. Importantly, the process still allows the operator to evaluate and have control over all the phases of quantification process.
CMA: chaperone-mediated autophagy; CSV: comma separated values; eMI: endosomal microautophagy; Fiji: Fiji is just ImageJ; MA: macroautophagy; SParQ: Streamlined Particle Quantification.
内溶酶体系统对于细胞内蛋白质的动态平衡至关重要。研究蛋白质运输和降解的常用方法(例如通过自噬)是通过用荧光标签或荧光抗体染色标记囊泡结构,如内体、自噬体、溶酶体或模型底物。详细分析需要在各种条件下对数百个结构进行定量。通常,使用广泛可用的 Fiji/ImageJ(https://imagej.net/Fiji/Downloads)等软件逐个分析图像,独立调整和阈值化每个图像和通道,这是一项非常耗时且繁琐的任务。为了简化该过程,我们开发了一个插件,该插件集成到 Fiji 中,可实现囊泡(即点状)结构的自动定量。重要的是,该过程仍然允许操作人员评估和控制定量过程的所有阶段。
CMA:伴侣介导的自噬;CSV:逗号分隔值;eMI:内体微自噬;Fiji:Fiji 就是 ImageJ;MA:巨自噬;SParQ:简化粒子定量。