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Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control


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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Figure 1: PSF schematics and theoretical functions.
Figure 2: Microsphere images and isosurfaces from a confocal microscope.
Figure 3: Lens cleaning protocol and pinhole and laser alignment.
Figure 4: Image acquisition settings to avoid data clipping.
Figure 5: Over- and under-sampling when measuring the PSF.
Figure 6: Using region tools to image single microspheres.
Figure 7: Ideal and distorted PSF measurements.
Figure 8: MetroloJ report summary.
Figure 9: PSF variability.


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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.

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Authors and Affiliations



R.W.C. and C.M.B. conceived of the need for a protocol for testing confocal microscope resolution, confocal microscope quality and objective lens quality. C.M.B. and R.W.C. prepared samples, collected images, analyzed images and interpreted data results. C.M.B. wrote the manuscript and designed the figures with critical reading and editing provided by R.W.C. T.J. collected PSF images, analyzed them and assimilated the data for the time-dependent studies.

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Correspondence to Claire M Brown.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Alternative procedure steps 28-47 to use if using the Zeiss 710, Zeiss 510, Olympus FV1000, Nikon A1 or Leica SP5 (PDF 1635 kb)

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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).

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