Abstract

Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an objective and quantitative manner. In this work, a method based on Fourier transform formalism is presented to analyze the performance of a STED system. The spatial frequency distribution of pairs of confocal and STED images are compared to obtain an objective parameter, the Azimuth Averaged Spectral Content Spread (AASCS), that is related to the performance of the system in which the sample is also considered. The method has been first tested on samples of beads, and then applied to cell samples labeled with multiple fluorescent dyes. The results show that a single parameter, the AASCS, can be used to determine the optimal settings for STED image acquisition in an objective way, only by using the information provided by the images from the sample themselves. The AASCS also helps minimize the depletion power, for better preservation of the samples.

© 2017 Optical Society of America

Full Article  |  PDF Article
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References

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  27. G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
    [Crossref] [PubMed]

2016 (2)

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
[Crossref] [PubMed]

L. Frahm and J. Keller, “Polarization modulation adds little additional information to super-resolution fluorescence microscopy,” Nat. Methods 13(1), 7–8 (2016).
[Crossref] [PubMed]

2015 (2)

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

J. Tam and D. Merino, “Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods,” J. Neurochem. 135(4), 643–658 (2015).
[Crossref] [PubMed]

2014 (3)

P. Theer, C. Mongis, and M. Knop, “PSFj: know your fluorescence microscope,” Nat. Methods 11(10), 981–982 (2014).
[Crossref] [PubMed]

C. Tressler, M. Stolle, and C. Fradin, “Fluorescence correlation spectroscopy with a doughnut-shaped excitation profile as a characterization tool in STED microscopy,” Opt. Express 22(25), 31154–31166 (2014).
[Crossref] [PubMed]

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

2013 (2)

K. I. Hng and D. Dormann, “ConfocalCheck--a software tool for the automated monitoring of confocal microscope performance,” PLoS One 8(11), e79879 (2013).
[Crossref] [PubMed]

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

2012 (2)

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express 20(5), 5225–5236 (2012).
[Crossref] [PubMed]

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

2011 (4)

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 6(12), 1929–1941 (2011).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

2010 (2)

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

2009 (1)

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[Crossref]

2008 (1)

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

2007 (2)

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

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

2006 (1)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

2003 (1)

2000 (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[Crossref] [PubMed]

1999 (1)

1998 (1)

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[Crossref] [PubMed]

1994 (1)

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Bates, M.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Beater, S.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Berning, S.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

Bewersdorf, J.

Booth, M. J.

Brown, C. M.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 6(12), 1929–1941 (2011).
[Crossref] [PubMed]

Bückers, J.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Burke, D.

Clouvel, G.

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

Cole, R. W.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 6(12), 1929–1941 (2011).
[Crossref] [PubMed]

Colón-Ramos, D. A.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Davis, M. W.

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Dedecker, P.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Dibaj, P.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

Donnert, G.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Dormann, D.

K. I. Hng and D. Dormann, “ConfocalCheck--a software tool for the automated monitoring of confocal microscope performance,” PLoS One 8(11), e79879 (2013).
[Crossref] [PubMed]

Ducommun, B.

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

Dyba, M.

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[Crossref] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[Crossref] [PubMed]

Eggeling, C.

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express 20(5), 5225–5236 (2012).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[Crossref]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[Crossref] [PubMed]

Egner, A.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[Crossref] [PubMed]

Engelhardt, J.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

Escande, P.

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

Fradin, C.

Frahm, L.

L. Frahm and J. Keller, “Polarization modulation adds little additional information to super-resolution fluorescence microscopy,” Nat. Methods 13(1), 7–8 (2016).
[Crossref] [PubMed]

Fron, E.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Frongia, C.

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

Goodwin, P. C.

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

Gould, T. J.

Grünwald, D.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Han, K. Y.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[Crossref]

Harke, B.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Hell, S. W.

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express 20(5), 5225–5236 (2012).
[Crossref] [PubMed]

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[Crossref]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[Crossref] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[Crossref] [PubMed]

T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
[Crossref] [PubMed]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[Crossref] [PubMed]

Hng, K. I.

K. I. Hng and D. Dormann, “ConfocalCheck--a software tool for the automated monitoring of confocal microscope performance,” PLoS One 8(11), e79879 (2013).
[Crossref] [PubMed]

Hobson, R. J.

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Hofkens, J.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Hollopeter, G.

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Holzmeister, P.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Hotta, J.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Irvine, S. E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[Crossref]

Jahn, R.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Jakobs, S.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[Crossref] [PubMed]

Janssen, K. P. F.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Jinadasa, T.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 6(12), 1929–1941 (2011).
[Crossref] [PubMed]

Jorgensen, E. M.

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Keller, J.

L. Frahm and J. Keller, “Polarization modulation adds little additional information to super-resolution fluorescence microscopy,” Nat. Methods 13(1), 7–8 (2016).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[Crossref] [PubMed]

T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
[Crossref] [PubMed]

Knop, M.

P. Theer, C. Mongis, and M. Knop, “PSFj: know your fluorescence microscope,” Nat. Methods 11(10), 981–982 (2014).
[Crossref] [PubMed]

Lalkens, B.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Leutenegger, M.

Li, C.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Lidke, K. A.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Lorenzo, C.

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Masson, A.

A. Masson, P. Escande, C. Frongia, G. Clouvel, B. Ducommun, and C. Lorenzo, “High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM,” Sci. Rep. 5, 16898 (2015).
[Crossref] [PubMed]

Medda, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Merino, D.

J. Tam and D. Merino, “Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods,” J. Neurochem. 135(4), 643–658 (2015).
[Crossref] [PubMed]

Moneron, G.

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express 20(5), 5225–5236 (2012).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Mongis, C.

P. Theer, C. Mongis, and M. Knop, “PSFj: know your fluorescence microscope,” Nat. Methods 11(10), 981–982 (2014).
[Crossref] [PubMed]

Müllen, K.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

Nelson, J. C.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Nieuwenhuizen, R. P. J.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Owald, D.

Patton, B. R.

Pibiri, E.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Puig, D. L.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Punge, A.

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Rankin, B. R.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Reuss, M.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

Rieger, B.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Rigler, R.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[Crossref] [PubMed]

Rittweger, E.

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express 20(5), 5225–5236 (2012).
[Crossref] [PubMed]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[Crossref]

Rizzoli, S. O.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[Crossref] [PubMed]

Schmidt, R.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

Schönle, A.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

Schroeder, J.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Schwarzer, D.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Seidel, C. A. M.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[Crossref] [PubMed]

Stallinga, S.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Steffens, H.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

Stolle, M.

Ta, H.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

Tam, J.

J. Tam and D. Merino, “Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods,” J. Neurochem. 135(4), 643–658 (2015).
[Crossref] [PubMed]

Theer, P.

P. Theer, C. Mongis, and M. Knop, “PSFj: know your fluorescence microscope,” Nat. Methods 11(10), 981–982 (2014).
[Crossref] [PubMed]

Tinnefeld, P.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Tressler, C.

Vicidomini, G.

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express 20(5), 5225–5236 (2012).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

Walter, A.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

Watanabe, S.

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Westphal, V.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

Wichmann, J.

Widengren, J.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[Crossref] [PubMed]

Willig, K. I.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, S. W. Hell, and E. M. Jorgensen, “Protein localization in electron micrographs using fluorescence nanoscopy,” Nat. Methods 8(1), 80–84 (2011).
[Crossref] [PubMed]

Wurm, C. A.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

Anal. Chem. (1)

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[Crossref] [PubMed]

Appl. Opt. (1)

Biophys. J. (2)

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[Crossref] [PubMed]

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[Crossref] [PubMed]

J. Am. Chem. Soc. (1)

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[Crossref] [PubMed]

J. Neurochem. (1)

J. Tam and D. Merino, “Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods,” J. Neurochem. 135(4), 643–658 (2015).
[Crossref] [PubMed]

Methods Cell Biol. (1)

P. C. Goodwin, “Evaluating optical aberration using fluorescent microspheres: methods, analysis, and corrective actions,” Methods Cell Biol. 81, 397–413 (2007).
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Nat. Methods (6)

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Figures (5)

Fig. 1
Fig. 1

Confocal (a) and STED (b) images of the same sample of Chromeo 488 beads (70nm diameter). Scale bar is 5µm. (c) Radial average of the FT of the confocal and STED images shown in a) and b). (d) The data in c) has been normalized following the procedure described in this article. (e) Difference between the normalized data for the confocal and STED images. The difference is maximum on the spatial frequencies around the diffraction limit, which is shown in the plots as a vertical dotted line, the value of this difference is a measure of the contrast improvement.

Fig. 2
Fig. 2

Variation of the AASCS with STED laser beam power (in arbitrary units) for images of Chromeo 488 beads.

Fig. 3
Fig. 3

a) FT of confocal images of beads averaged around the azimuth, showing the reduction in noise level as excitation laser power increases. b) Width of the Gaussian function fitting the AASC for confocal images of beads acquired at excitation laser powers of 2, 4, 6, 8 and 10%. Higher values of the excitation laser power produced saturation on the image and were discarded.

Fig. 4
Fig. 4

(a) Confocal image of a HeLa cell sample where endoplasmic reticulum has been tagged with BD Horizon V500 (green) and secretory vesicles with Oregon Green 488 (red). Image is 30.4x30.4µm in size. (b) and (c) are smaller sections of the image in a) acquired with confocal, b), and STED, c). (d) Azimuth average of the FT of the confocal and STED images in b) and c) for the BD Horizon V500 channel. Green markers show the data of the STED images, and orange the confocal ones. Corresponding fits of the data (dotted line for STED and solid for confocal) using the method described are also shown in the plots. (e) Similar to d) for the Oregon Green 488 channel. (f) AASCS calculated for the BD Horizon V500 and Oregon Green 488 channels of the HeLa samples described. The maximum AASCS is achieved at 60% depletion laser power for the Oregon Green 488, and 40% in the case of the BD Horizon V500.

Fig. 5
Fig. 5

(a-f) Confocal and STED pairs of images from HeLa cell samples where secretory vesicles have been tagged with Oregon Green 488. A gated STED system from Leica was used on this experiment. STED images have been acquired setting the depletion laser power to 20%, b), 60%, d) and 100%, f). The images are acquired using photon counting settings, and the confocal (a,c, and e) and STED (b, d and f) images are shown using the same color scale to minimize display effects. (g) Azimuth average of the FT of the confocal and STED images in c) and d). Green markers show the data from the STED images, and orange from the confocal ones. Corresponding fits of the data using the method described are also shown in the plots, dotted line for STED and solid for confocal. (h) AASCS obtained using different excitation powers in the STED laser on the same sample. The data have been calculated using the images a-f), amongst others. The AASCS is maximum for values of the depletion laser power of around 60%.

Equations (6)

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I(x,y)=O( x,y )PSF(x,y)
I ˜ (u,v)=FT{ O( x,y ) }FT{ PSF(x,y) }= O ˜ (u,v)OTF(u,v)
I(x,y)= i I bead (x x i ,y y i ) +N(x,y)
I ˜ (u,v)= I ˜ bead (u,v) i C i e -j x i u e j y i v + N ˜ (u,v)= = O ˜ bead (u,v)·OTF(u,v) i C i e -j x i u e j y i v + N ˜ (u,v)
I ˜ (k,φ)= O ˜ bead (k)·OTF(k) i C i e -jk x i cosφ e jk y i sinφ + N ˜ (k,φ)
log[ I ˜ (k) ]AB e ( ( k k 0 ) 2 2 σ 2 ) +C e ( λ| k k 0 | ) +D| ( k k 0 ) |

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