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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.


  1. 1

    Pawley, J.B. Handbook of Biological Confocal Microscopy 2nd ed. (Plenum Press, 1995).

  2. 2

    Goodwin, P.C. Evaluating optical aberration using fluorescent microspheres: methods, analysis, and corrective actions. Methods Cell Biol. 81, 397–413 (2007).

    Article  Google Scholar 

  3. 3

    Airy, G.B. On the diffraction of an object-glass with circular aperture. Trans. of the Cambridge Philosoph. Soc. 5, 283–291 (1835).

    Google Scholar 

  4. 4

    Wilhelm, S., Gröbler, B., Gluch, M. & Heinz, H. in Carl Zeiss Principles (Carl Zeiss, 1997).

  5. 5

    Beyer, H. Handbuch der Mikroskopie 2nd ed. (VEB Verlag Technik, 1985).

  6. 6

    Gibson, S.F. & Lanni, F. Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy. J. Opt. Soc. Am. A 9, 154–166 (1992).

    CAS  Article  Google Scholar 

  7. 7

    Hiraoka, Y., Sedat, J.W. & Agard, D.A. Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy. Biophys. J. 57, 325–333 (1990).

    CAS  Article  Google Scholar 

  8. 8

    Goodwin, P.C. in Digital Microscopy 3rd ed., Vol. 81 (eds. Sluder, G. & Wolf, D.E.) 397–413 (Elsevier, 2007).

  9. 9

    Brown, C.M. et al. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J. Microsc. 229, 78–91 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Digman, M.A., Brown, C.M., Horwitz, A.R., Mantulin, W.W. & Gratton, E. Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy. Biophys. J. 94, 2819–2831 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Digman, M.A. et al. Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys. J. 89, 1317–1327 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Digman, M.A. et al. Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys. J. 88, L33–L36 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Wiseman, P.W. et al. Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy. J. Cell Sci. 117, 5521–5534 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Wallace, W., Schaefer, L.H. & Swedlow, J.R. A workingperson's guide to deconvolution in light microscopy. Biotechniques 31, 1076–1078, 1080, 1082 passim (2001).

    CAS  Article  Google Scholar 

  15. 15

    Swedlow, J.R. Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol. 81, 447–465 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Swedlow, J.R. Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol. 72, 349–367 (2003).

    Article  Google Scholar 

  17. 17

    Swedlow, J.R. & Platani, M. Live cell imaging using wide-field microscopy and deconvolution. Cell Struct. Funct. 27, 335–341 (2002).

    Article  Google Scholar 

  18. 18

    Cole, R.W. & Turner, J.N. Light-emitting diodes are better illumination sources for biological microscopy than conventional sources. Microsc. Microanal. 14, 243–250 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Lacoste, J., Vining, C., Zuo, D., Spurmanis, A. & Brown, C.M. Optimal conditions for live-cell microscopy and raster image correlation spectroscopy (RICS). in Annual Reviews in Fluorescence 2010 (ed. Geddes, C.D.) (2011).

  20. 20

    Koehler, A. New method of illumination for photomicrographical purposes. J. Royal Microscopical Soc. 14, 261–262 (1894).

    Google Scholar 

  21. 21

    Nyquist, H. Certain topics in telegraph transmission theory. Trans. AIEE 46, 617–644 (1928).

    Google Scholar 

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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.

Author information




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.

Corresponding author

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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