Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena.
Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena.
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发表时间：
1991-06
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通讯作者：
Yasushi Hiraoka;Swedlow;M. Paddy;D. Agard;J. Sedat
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作者：
Yasushi Hiraoka;Swedlow;M. Paddy;D. Agard;J. Sedat
细胞事件是由细胞内三维环境中细胞组分的协调相互作用完成的。在三维空间中同时观察多个组分对于理解这种相互作用至关重要。为此，我们开发了一种计算机显微镜工作站，能够记录固定细胞和活细胞中多种细胞成分的三维图像。显微镜控制，数据采集，图像处理和分析的所有方面都可以在一个工作站上完成。在本报告中，我们描述了这个集成系统的组件和功能。此外，我们还讨论了多波长、三维成像的一些一般问题，以及该技术在果蝇染色体组织分析中的应用。间接免疫荧光染色的固定胚胎三维成像显示了染色体、微管和核层的结构组织。注射荧光标记蛋白的活胚胎成像证实并扩展了这些结果，允许对整个细胞周期的这些结构进行研究。荧光显微镜的分子特异性和我们工作站获得的三维结构信息的结合，为细胞周期中染色体行为的动态方面提供了新的见解。我们相信这个系统在研究细胞事件的分子基础方面有许多重要的应用。
Cellular events are accomplished by the coordinated interactions of cellular components within the three-dimensional context of a cell. Simultaneous observation of multiple components in three dimensions can be essential for understanding such interactions. Toward this end, we have developed a computerized microscope workstation capable of recording three-dimensional images of multiple cellular components in fixed and living cells. All aspects of microscope control, data collection, image processing and analysis can be performed on the one workstation. In this report, we describe the components and capabilities of this integrated system. In addition, we discuss some general problems of multiple-wavelength, three-dimensional imaging and our application of this technology to the analysis of chromosome organization in Drosophila melanogaster. Three-dimensional imaging of fixed embryos stained by indirect immunofluorescence has revealed the structural organization of chromosomes, microtubules, and the nuclear lamins. Imaging of living embryos injected with fluorescently labelled proteins has confirmed and extended these results by allowing the study of these structures throughout the cell cycle. The combination of the molecular specificity of fluorescence microscopy and the three-dimensional structural information obtained by our workstation has provided novel insights into the dynamic aspects of chromosome behavior during the cell cycle. We believe this system has many important applications in the study of the molecular basis of cellular events.