Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts.
Division of Medical Sciences, Harvard Medical School, Boston, Massachusetts.
Curr Protoc Cell Biol. 2020 Jun;87(1):e108. doi: 10.1002/cpcb.108.
Trafficking of intracellular cargo is essential to cellular function and can be defective in pathological states including cancer and neurodegeneration. Tools to quantify intracellular traffic are thus necessary for understanding this fundamental cellular process, studying disease mechanisms, and testing the effects of therapeutic pharmaceuticals. In this article we introduce an algorithm called QuoVadoPro that autonomously quantifies the movement of fluorescently tagged intracellular cargo. QuoVadoPro infers the extent of intracellular motility based on the variance of pixel illumination in a series of time-lapse images. The algorithm is an unconventional approach to the automatic measurement of intracellular traffic and is suitable for quantifying movements of intracellular cargo under diverse experimental paradigms. QuoVadoPro is particularly useful to measure intracellular cargo movement in non-neuronal cells, where cargo trafficking occurs as short movements in mixed directions. The algorithm can be applied to images with low temporal or spatial resolutions and to intracellular cargo with varying shapes or sizes, like mitochondria or endoplasmic reticulum: situations in which conventional methods such as kymography and particle tracking cannot be applied. In this article we present a stepwise protocol for using the QuoVadoPro software, illustrate its methodology with common examples, discuss critical parameters for reliable data analysis, and demonstrate its use with a previously published example. © 2020 Wiley Periodicals LLC. Basic Protocol: QuoVadoPro, an autonomous tool for measuring intracellular dynamics using temporal variance.
细胞内货物的运输对于细胞功能至关重要，在包括癌症和神经退行性疾病在内的病理状态下可能会出现缺陷。因此，用于量化细胞内运输的工具对于理解这一基本细胞过程、研究疾病机制以及测试治疗药物的效果是必要的。在本文中，我们介绍了一种名为 QuoVadoPro 的算法，该算法可以自主量化荧光标记的细胞内货物的运动。QuoVadoPro 根据一系列时程图像中像素照明的方差来推断细胞内运动的程度。该算法是自动测量细胞内运输的一种非传统方法，适用于量化各种实验范式下细胞内货物的运动。QuoVadoPro 特别适用于测量非神经元细胞中的细胞内货物运动，在这些细胞中，货物运输表现为混合方向的短距离运动。该算法可应用于时间或空间分辨率较低的图像，以及具有不同形状或大小的细胞内货物，如线粒体或内质网：在这些情况下，传统方法（如动图和粒子追踪）无法应用。本文介绍了使用 QuoVadoPro 软件的分步协议，用常见示例说明了其方法，讨论了可靠数据分析的关键参数，并通过一个先前发表的示例演示了其用途。© 2020 威立出版公司。基本方案：QuoVadoPro，一种使用时间方差测量细胞内动态的自主工具。
