Single-cell quantification of molecules and rates using open-source microscope-based cytometry
Single-cell quantification of molecules and rates using open-source microscope-based cytometry
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DOI：
10.1038/nmeth1008
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发表时间：
2007-02-01
期刊：
影响因子：
48
通讯作者：
Brent, Roger
中科院分区：
文献类型：
--
作者：
Gordon, Andrew;Colman-Lerner, Alejandro;Brent, Roger
基于显微镜的细胞术为高通量研究细胞提供了强有力的手段。在这里，我们提出了一套完善的方法，使大量的个体酿酒酵母细胞随着时间的推移进行灵敏的测量。这套方法包括相对简单的“湿”方法、显微镜程序、开源软件工具和统计程序。这种组合非常灵敏，允许检测和测量每个活酵母细胞少于350个荧光蛋白分子。这些方法使新的协议，包括“快照”协议，以计算成熟和降解的分子种类，包括GFP衍生物和天然mRNA，在未受干扰的，指数增长的酵母细胞的速率。由于它们的灵敏度，准确性和跟踪单个细胞随时间变化的能力，这些显微镜方法可以补充流式细胞术测量细胞系统的定量生理学研究。
Microscope-based cytometry provides a powerful means to study cells in high throughput. Here we present a set of refined methods for making sensitive measurements of large numbers of individual Saccharomyces cerevisiae cells over time. The set consists of relatively simple 'wet' methods, microscope procedures, open-source software tools and statistical routines. This combination is very sensitive, allowing detection and measurement of fewer than 350 fluorescent protein molecules per living yeast cell. These methods enabled new protocols, including 'snapshot' protocols to calculate rates of maturation and degradation of molecular species, including a GFP derivative and a native mRNA, in unperturbed, exponentially growing yeast cells. Owing to their sensitivity, accuracy and ability to track changes in individual cells over time, these microscope methods may complement flow-cytometric measurements for studies of the quantitative physiology of cellular systems.