This protocol outlines a procedure for collecting and analyzing point spread functions (PSFs). It describes how to prepare fluorescent microsphere samples, set up a confocal microscope to properly collect 3D confocal image data of the microspheres and perform PSF measurements. The analysis of the PSF is used to determine the resolution of the microscope and to identify any problems with the quality of the microscope's images. The PSF geometry is used as an indicator to identify problems with the objective lens, confocal laser scanning components and other relay optics. Identification of possible causes of PSF abnormalities and solutions to improve microscope performance are provided. The microsphere sample preparation requires 2–3 h plus an overnight drying period. The microscope setup requires 2 h (1 h for laser warm up), whereas collecting and analyzing the PSF images require an additional 2–3 h.
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We acknowledge the use of the Wadsworth Center's Advanced Light Microscopy & Image Analysis Core Facility, as well as the McGill Life Sciences Complex Imaging Facility for portions of the work presented. We thank R. Stack for help with sample preparation, PSF image stack acquisition and data analysis. We thank B. Northan from Media Cybernetics for generating the theoretical PSF images for Figure 1, C. Glowinski for preparing 3D PSF figures using the Bitplane Imaris software for Figure 2, A. Spurmanis for providing objective lens cleaning images for Figure 3, as well as F. Waharte and M. Thibault for providing Supplementary Methods on Nikon and Olympus confocals, respectively. We thank B. Eason and K. Young for critical reading of the manuscript.
The authors declare no competing financial interests.
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Cole, R., Jinadasa, T. & Brown, C. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nat Protoc 6, 1929–1941 (2011). https://doi.org/10.1038/nprot.2011.407
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