Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening
Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening
复制标题
DOI：
10.1074/mcp.t700006-mcp200
复制
发表时间：
2007-08-01
影响因子：
7
通讯作者：
Wouters, Fred S.
中科院分区：
文献类型：
--
作者：
Esposito, Alessandro;Dohm, Christoph P.;Wouters, Fred S.
蛋白质组学和细胞组学显然受益于细胞生化事件的分子洞察，这些洞察可以通过先进的定量显微镜技术（如荧光寿命成像显微镜和福斯特共振能量转移成像）获得。在分子水平检测到的光谱信息可以与细胞形态估计器、细胞定位分析以及分子或细胞亚群的识别相结合。这使得创建强大的检测方法能够详细了解细胞对化学和物理刺激的时空反应的分子机制。这项工作表明，这些技术提供的高含量可以与能够进行无人监督操作和图像分析的荧光寿命成像显微镜设置自动化提供的高通量水平相结合。专用于图像细胞术分析和分类的系统和软件代表了蛋白质组学、相互作用组学和细胞组学领域的重要新兴工具。通过应用新的全固态技术，这些技术很快就会为学术界和药物筛选界提供，这可能会产生具有成本效益的统包系统。这里展示了这种筛选技术在细胞内 α-突触核蛋白泛素化水平及其导致帕金森病的家族突变的研究中的应用。统计上突变型 α-突触核蛋白形式的泛素化程度较低的发现支持了这种修饰在病理性蛋白质聚集机制中的作用。
Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Forster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.