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Tutorial: guidance for quantitative confocal microscopy.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-03-31 , DOI: 10.1038/s41596-020-0313-9 James Jonkman 1 , Claire M Brown 2 , Graham D Wright 3 , Kurt I Anderson 4 , Alison J North 5
Nature Protocols ( IF 13.1 ) Pub Date : 2020-03-31 , DOI: 10.1038/s41596-020-0313-9 James Jonkman 1 , Claire M Brown 2 , Graham D Wright 3 , Kurt I Anderson 4 , Alison J North 5
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When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, choosing the most suitable microscope for a given application and configuring the microscope parameters. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. Common pitfalls such as photobleaching and cross-talk are addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. Finally, guidelines for analyzing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. A visual summary of this tutorial is available as a poster (https://doi.org/10.1038/s41596-020-0307-7).
中文翻译：
教程：定量共聚焦显微镜指南。
如果使用得当，共聚焦荧光显微镜是对细胞和组织进行定量测量的绝佳工具。共聚焦显微镜能够阻挡离焦光，从而对样品进行光学切片，使研究人员能够以非常高的空间精度量化荧光。然而，使用共聚焦显微镜生成有意义的数据需要仔细规划和对该技术的透彻理解。在本教程中，研究人员将了解获取定量共聚焦显微镜图像的各个方面，包括优化固定细胞和活细胞的样品制备、为给定应用选择最合适的显微镜以及配置显微镜参数。为规划公正和严格的共焦显微镜实验提供了建议。解决了常见的缺陷，例如光漂白和串扰，以及可能阻止定量数据采集的几个令人不安的仪器问题。最后，提出了以保持数据的定量性质的方式分析和呈现共焦图像的指南，并讨论了统计分析。本教程的可视化摘要以海报形式提供 (https://doi.org/10.1038/s41596-020-0307-7)。
更新日期：2020-03-31
中文翻译：
教程：定量共聚焦显微镜指南。
如果使用得当，共聚焦荧光显微镜是对细胞和组织进行定量测量的绝佳工具。共聚焦显微镜能够阻挡离焦光，从而对样品进行光学切片，使研究人员能够以非常高的空间精度量化荧光。然而，使用共聚焦显微镜生成有意义的数据需要仔细规划和对该技术的透彻理解。在本教程中，研究人员将了解获取定量共聚焦显微镜图像的各个方面，包括优化固定细胞和活细胞的样品制备、为给定应用选择最合适的显微镜以及配置显微镜参数。为规划公正和严格的共焦显微镜实验提供了建议。解决了常见的缺陷，例如光漂白和串扰，以及可能阻止定量数据采集的几个令人不安的仪器问题。最后，提出了以保持数据的定量性质的方式分析和呈现共焦图像的指南，并讨论了统计分析。本教程的可视化摘要以海报形式提供 (https://doi.org/10.1038/s41596-020-0307-7)。
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