Advanced Optical Microscopy Facility (AOMF), University Health Network, Toronto, Ontario, Canada.
Advanced BioImaging Facility (ABIF), McGill University, Montreal, Quebec, Canada.
Nat Protoc. 2020 May;15(5):1585-1611. doi: 10.1038/s41596-020-0313-9. Epub 2020 Mar 31.
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).
当适当地使用时，共聚焦荧光显微镜是对细胞和组织进行定量测量的极好工具。共聚焦显微镜能够阻挡离焦的光，从而通过标本进行光学切片，使研究人员能够以非常高的空间精度对荧光进行定量。然而，要使用共聚焦显微镜生成有意义的数据，需要仔细规划并充分了解该技术。在本教程中，研究人员将通过获取定量共聚焦显微镜图像的所有方面进行指导，包括优化固定和活细胞的样品制备、为特定应用选择最合适的显微镜以及配置显微镜参数。还为无偏和严格的共聚焦显微镜实验提供了规划建议。解决了诸如光漂白和串扰等常见陷阱，以及可能阻止获取定量数据的几个麻烦的仪器问题。最后，介绍了以保持数据定量性质的方式分析和呈现共聚焦图像的指南，并讨论了统计分析。本教程的可视化摘要可作为海报获取（https://doi.org/10.1038/s41596-020-0307-7）。
