Abstract

Precise knowledge of the effective spatial resolution in a stimulated emission depletion (STED) microscopy experiment is essential for reliable interpretation of the imaging results. STED microscopy theoretically provides molecular resolution, but practically different factors limit its resolution. Because these factors are related to both the sample and the system, a reliable estimation of the resolution is not straightforward. Here we show a method based on the Fourier ring correlation (FRC), which estimates an absolute resolution value directly from any STED and, more in general, point-scanning microscopy image. The FRC-based resolution metric shows terrific sensitivity to the image signal-to-noise ratio, as well as to all sample and system dependent factors. We validated the method both on commercial and on custom-made microscopes. Since the FRC-based metric can be computed in real time, without any prior information of the system/sample, it can become a fundamental tool for (i) microscopy users to optimize the experimental conditions and (ii) microscopy specialists to optimize the system conditions.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Full Article  |  PDF Article
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References

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D. Merino, A. Mallabiabarrena, J. Andilla, D. Artigas, T. Zimmermann, and P. Loza-Alvarez, Biomed. Opt. Express 8, 2472 (2017).
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2016 (4)

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, Opt. Express 24, 8862 (2016).
[Crossref]

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

D. Li and E. Betzig, Science 352, 527 (2016).
[Crossref]

2015 (1)

C. Eggeling, K. I. Willig, S. J. Sahl, and S. W. Hell, Q. Rev. Biophys. 48, 178 (2015).
[Crossref]

2013 (5)

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, J. Struct. Biol. 183, 363 (2013).
[Crossref]

T. J. Gould, E. B. Kromann, D. Burke, M. J. Booth, and J. Bewersdorf, Opt. Lett. 38, 1860 (2013).
[Crossref]

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

R. Zanella, G. Zanghirati, R. Cavicchioli, L. Zanni, P. Boccacci, M. Bertero, and G. Vicidomini, Sci. Rep. 3, 2523 (2013).
[Crossref]

2012 (2)

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, Opt. Express 20, 5225 (2012).
[Crossref]

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

2010 (1)

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

2005 (1)

V. Westphal and S. W. Hell, Phys. Rev. Lett. 94, 143903 (2005).
[Crossref]

1994 (1)

1987 (2)

M. van Heel, Ultramicroscopy 21, 95 (1987).
[Crossref]

M. Unser, B. L. Trus, and A. C. Steven, Ultramicroscopy 23, 39 (1987).
[Crossref]

1982 (1)

W. O. Saxton and W. Baumeister, J. Microsc. 127, 127 (1982).
[Crossref]

Andilla, J.

Artigas, D.

Balzarotti, F.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

Banterle, N.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, J. Struct. Biol. 183, 363 (2013).
[Crossref]

Bates, M.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Baumeister, W.

W. O. Saxton and W. Baumeister, J. Microsc. 127, 127 (1982).
[Crossref]

Beck, M.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, J. Struct. Biol. 183, 363 (2013).
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Bertero, M.

R. Zanella, G. Zanghirati, R. Cavicchioli, L. Zanni, P. Boccacci, M. Bertero, and G. Vicidomini, Sci. Rep. 3, 2523 (2013).
[Crossref]

Betzig, E.

D. Li and E. Betzig, Science 352, 527 (2016).
[Crossref]

Bewersdorf, J.

Bianchini, A. D.

G. Vicidomini and A. D. Bianchini, “STED super-resolved microscopy,” Nat. Methods (to be published).

Bianchini, P.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Boccacci, P.

R. Zanella, G. Zanghirati, R. Cavicchioli, L. Zanni, P. Boccacci, M. Bertero, and G. Vicidomini, Sci. Rep. 3, 2523 (2013).
[Crossref]

Booth, M. J.

Boso, G.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Bückers, J.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Bui, K. H.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, J. Struct. Biol. 183, 363 (2013).
[Crossref]

Burke, D.

Buttafava, M.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Castello, M.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Cavicchioli, R.

R. Zanella, G. Zanghirati, R. Cavicchioli, L. Zanni, P. Boccacci, M. Bertero, and G. Vicidomini, Sci. Rep. 3, 2523 (2013).
[Crossref]

Chmyrov, A.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
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Dammeyer, T.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
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Dedecker, P.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Diaspro, A.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Eggeling, C.

C. Eggeling, K. I. Willig, S. J. Sahl, and S. W. Hell, Q. Rev. Biophys. 48, 178 (2015).
[Crossref]

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, Opt. Express 20, 5225 (2012).
[Crossref]

Egner, A.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
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Forthmann, C.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

Fron, E.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Gietl, A.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

Gould, T. J.

Grotjohann, T.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

Grünwald, D.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Han, K. Y.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

Harke, B.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Hell, S. W.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, Sci. Rep. 7, 11354 (2017).
[Crossref]

S. J. Sahl, S. W. Hell, and S. Jakobs, Nat. Rev. Mol. Cell Biol. 18, 685 (2017).
[Crossref]

C. Eggeling, K. I. Willig, S. J. Sahl, and S. W. Hell, Q. Rev. Biophys. 48, 178 (2015).
[Crossref]

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, Opt. Express 20, 5225 (2012).
[Crossref]

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

V. Westphal and S. W. Hell, Phys. Rev. Lett. 94, 143903 (2005).
[Crossref]

S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994).
[Crossref]

Hernández, I. C.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Hofkens, J.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Holzmeister, P.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

Hotta, J.-I.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Jakobs, S.

S. J. Sahl, S. W. Hell, and S. Jakobs, Nat. Rev. Mol. Cell Biol. 18, 685 (2017).
[Crossref]

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

Janssen, K. P. F.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Keller-Findeisen, J.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

Kromann, E. B.

Lavoie-Cardinal, F.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

Lemke, E. A.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, J. Struct. Biol. 183, 363 (2013).
[Crossref]

Leutenegger, M.

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

Li, C.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Li, D.

D. Li and E. Betzig, Science 352, 527 (2016).
[Crossref]

Lidke, K.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Loza-Alvarez, P.

Mallabiabarrena, A.

Merino, D.

Moneron, G.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, Opt. Express 20, 5225 (2012).
[Crossref]

Müllen, K.

J.-I. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, J. Am. Chem. Soc. 132, 5021 (2010).
[Crossref]

Nieuwenhuizen, R.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Oracz, J.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, Sci. Rep. 7, 11354 (2017).
[Crossref]

Owald, D.

Patton, B. R.

Puig, D.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Radzewicz, C.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, Sci. Rep. 7, 11354 (2017).
[Crossref]

Rieger, B.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Rittweger, E.

Sahl, S. J.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, Sci. Rep. 7, 11354 (2017).
[Crossref]

S. J. Sahl, S. W. Hell, and S. Jakobs, Nat. Rev. Mol. Cell Biol. 18, 685 (2017).
[Crossref]

S. J. Sahl, F. Balzarotti, J. Keller-Findeisen, M. Leutenegger, V. Westphal, A. Egner, F. Lavoie-Cardinal, A. Chmyrov, T. Grotjohann, and S. Jakobs, Science 352, 527 (2016).
[Crossref]

C. Eggeling, K. I. Willig, S. J. Sahl, and S. W. Hell, Q. Rev. Biophys. 48, 178 (2015).
[Crossref]

Saxton, W. O.

W. O. Saxton and W. Baumeister, J. Microsc. 127, 127 (1982).
[Crossref]

Schmied, J. J.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

Schönle, A.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

Stallinga, S.

R. Nieuwenhuizen, K. Lidke, M. Bates, D. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Steinhauer, C.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

Steven, A. C.

M. Unser, B. L. Trus, and A. C. Steven, Ultramicroscopy 23, 39 (1987).
[Crossref]

Ta, H.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, PLoS ONE 8, e54421 (2013).
[Crossref]

Tinnefeld, P.

J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, and P. Tinnefeld, Nat. Methods 9, 1133 (2012).
[Crossref]

Tortarolo, G.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Tosi, A.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, Microsc. Res. Tech. 79, 785 (2016).
[Crossref]

Trus, B. L.

M. Unser, B. L. Trus, and A. C. Steven, Ultramicroscopy 23, 39 (1987).
[Crossref]

Unser, M.

M. Unser, B. L. Trus, and A. C. Steven, Ultramicroscopy 23, 39 (1987).
[Crossref]

van Heel, M.

M. van Heel, Ultramicroscopy 21, 95 (1987).
[Crossref]

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Supplementary Material (1)

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Figures (4)

Fig. 1.
Fig. 1. (a) Correlation between the Fourier transforms of the two independent images over the perimeter of the circle of radius q is calculated, resulting in a FRC curve indicating the decay of the correlation with spatial frequency. The image resolution is the inverse of the spatial frequency for which the FRC curve drops below the threshold 1/70.143, so a threshold value at q=9.2  μm1 is equivalent to 108 nm resolution (excitation beam power Pexc=420  nW and STED beam power PSTED=60  mW). (b) FRC curves and (c) corresponding effective resolution values for two fixed Pexc and increasing PSTED. Insets show magnified views (renormalized in signal intensity) of the marked area (a) of the analyzed images for different Pexc and PSTED combinations. Pixel dwell time, 50 μs; pixel size, 30 nm; image format, 500×500  pixels; scale bars, 1 μm.
Fig. 2.
Fig. 2. FRC curves for imaging of 60 nm crimson fluorescent beads at (a) fixed STED beam power PSTED=12  mW and increasing excitation beam powers Pexc and (b) fixed Pexc=560  nW and increasing PSTED. The comparison between the FRC- and FWHM-based resolution as function of (a) Pexc and (b) PSTED. Insets show portions of the analyzed images for different combinations of Pexc and PSTED. Pixel dwell time, 50 μs; pixel size, 40 nm; format, 1000×1000  pixels; scale bars, 1 μm.
Fig. 3.
Fig. 3. FRC analysis on imaging of two different nanoruler lengths (90 and 160 nm) for increasing PSTED and Pexc=750, 1800, and 3000 nW. Portions of the analysis images representing an isolated nanoruler are reported together with the FRC-based resolution. Pixel dwell time, 100 μs; pixel size, 20 nm; format, 500×500  pixels; scale bars, 100 nm.
Fig. 4.
Fig. 4. (a),(b) FRC curves, crimson beads (60 nm) images, and the focal intensity distribution for the STED (red) and excitation (green) beams for an aligned (left) and a misaligned (right) configuration. (c) Details of STED images obtained with increasing delay (Δt) between the excitation pulses and the depletion pulses for conventional (top) and time-gated detection (bottom), and (d) the related FRC curves. The temporal scheme of the pulses and the detection is reported. (e) FRC-based resolution analysis as a function of Δt for both the conventional and time-gated detection. (a),(b) PSTED=38  mW and (c)–(e) 24 mW. (a),(b) Pexc=330  nW and (c)–(e) 560 nW. Pixel dwell time, 50 μs; pixel size, (a),(b) 20 nm and (c)–(e) 40 nm; format, 1000×1000  pixels; scale bars, 1 μm.

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