A computational platform for robotized fluorescence microscopy (I): High-content image-based cell-cycle analysis
A computational platform for robotized fluorescence microscopy (I): High-content image-based cell-cycle analysis
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DOI：
10.1002/cyto.a.22266
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发表时间：
2013-04-01
期刊：
影响因子：
3.7
通讯作者：
Faretta, Mario
中科院分区：
文献类型：
--
作者：
Furia, Laura;Pelicci, Pier Giuseppe;Faretta, Mario
硬件自动化和软件开发已经允许通过将优化的统计显著性与荧光显微镜相关联来显著增加图像采集和分析的吞吐量。尽管荧光显微镜和流式细胞术（FCM）之间有许多共同点，为后者开发的大量应用程序在现代高分辨率成像技术中发现了相对较低的空间。为了填补这一空白，我们开发了一种名为A.M.I. CO的新型计算平台（自动显微镜图像细胞计数），用于定量分析宽视场和共焦机器人显微镜的图像。由于建立了染色方案和分析程序，我们能够概括许多FCM测定。特别是，我们专注于DNA含量的测量和重建的细胞周期的最佳参数。标准的自动化显微镜采用最高的光学分辨率（200 nm），和白光源使得有可能进行有效的多参数分析。DNA和蛋白质含量的测量与其细胞内空间分布的图像来源的信息进行了补充。值得注意的是，开发的工具在图像分析和采集之间建立了直接联系。因此，有可能根据明确的定量分布分离目标群体，并将其物理重新定位用于衍射限制数据采集。由于其灵活性和分析驱动的采集，A.M.I. CO.可以集成流式细胞术、图像流和激光扫描细胞术分析，提供具有以前无法达到的统计相关性的高分辨率细胞内分析。(c)2013年国际细胞计数促进会（International Society for Advancement of Cytometry）
Hardware automation and software development have allowed a dramatic increase of throughput in both acquisition and analysis of images by associating an optimized statistical significance with fluorescence microscopy. Despite the numerous common points between fluorescence microscopy and flow cytometry (FCM), the enormous amount of applications developed for the latter have found relatively low space among the modern high-resolution imaging techniques. With the aim to fulfill this gap, we developed a novel computational platform named A.M.I.CO. (Automated Microscopy for Image-Cytometry) for the quantitative analysis of images from widefield and confocal robotized microscopes. Thanks to the setting up of both staining protocols and analysis procedures, we were able to recapitulate many FCM assays. In particular, we focused on the measurement of DNA content and the reconstruction of cell-cycle profiles with optimal parameters. Standard automated microscopes were employed at the highest optical resolution (200 nm), and white-light sources made it possible to perform an efficient multiparameter analysis. DNA- and protein-content measurements were complemented with image-derived information on their intracellular spatial distribution. Notably, the developed tools create a direct link between image-analysis and acquisition. It is therefore possible to isolate target populations according to a definite quantitative profile, and to relocate physically them for diffraction-limited data acquisition. Thanks to its flexibility and analysis-driven acquisition, A.M.I.CO. can integrate flow, image-stream and laser-scanning cytometry analysis, providing high-resolution intracellular analysis with a previously unreached statistical relevance. (c) 2013 International Society for Advancement of Cytometry (c) 2012 International Society for Advancement of Cytometry