Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. The 39 steps: a cautionary tale of quantitative 3-D fluorescence microscopy. A visual summary of this tutorial is available as a poster ( ). 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. 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. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. 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. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. 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. When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues.
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