Jonkman J, Brown CM, Wright GD, Anderson KI, North AJ. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc. 2020 May;15(5):1585-1611.
Abstract:
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).
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Funding Info:
Princess Margaret Foundation for ongoing financial support of the AOMF. G.D.W. thanks A*STAR and the National Research Foundation’s Shared Infrastructure Support Grant for continued support of the A*STAR Microscopy Platform. K.I.A. thanks the Francis Crick Institute for their CALM support. A.J.N. thanks the Rockefeller University for its continued support of the Frits and Rita Markus Bio-Imaging Resource Center (BIRC), the Sohn Conference Foundation for funding the Leica SP8 confocal microscope used to generate Figs. 3 and 4.