Abstract

In super-resolution localization microscopy, a final super-resolution image is built upon a huge amount of raw image frames (typically >10,000). To guarantee the quality of a final super-resolution image, researchers tend to collect as many raw images as possible, leading to excess data volume, which causes a significant waste of data acquisition time and computation resources. Here we present the structure-resolving index (SRI), an efficient criterion to end image acquisition automatically. Simulated and experimental data show that SRI can be used as a superior ending criterion to minimize the acquisition of excess data volume while ensuring the resolving ability of a final super-resolution image.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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2018 (2)

Y. M. Sigal, R. Zhou, and X. Zhuang, Science 361, 880 (2018).
[Crossref]

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

2017 (3)

Y. Wang, S. Jia, H. F. Zhang, D. Kim, H. Babcock, X. Zhuang, and L. Ying, Optica 4, 1277 (2017).
[Crossref]

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Z. Zhang, Y. Nishimura, and P. Kanchanawong, Mol. Biol. Cell 28, 333 (2017).
[Crossref]

2015 (2)

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

2013 (3)

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

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

2011 (3)

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, Nat. Methods 8, 499 (2011).
[Crossref]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, Nat. Methods 8, 1027 (2011).
[Crossref]

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

2010 (2)

Babcock, H.

Baird, M. A.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Bakalar, M.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

Bates, M.

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

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, Nat. Methods 8, 1027 (2011).
[Crossref]

Beghin, A.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Bewersdorf, J.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Briggs, J. A.

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

Burn, G. L.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Butler, C.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Cabillic, M.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Chen, B.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

Chen, K. H.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, Nat. Methods 8, 1027 (2011).
[Crossref]

Choquet, D.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Cope, A. P.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Cordes, V. C.

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

Daigle, N.

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

Davidson, M. W.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

de Marco, A.

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

Dempsey, G. T.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, Nat. Methods 8, 1027 (2011).
[Crossref]

Duim, W. C.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Ellenberg, J.

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

Galland, R.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Giannone, G.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Griffié, J.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Grünwald, D.

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

Hartwich, T. M.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

He, J.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, Nat. Methods 8, 499 (2011).
[Crossref]

Heard, N. A.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Hedde, P. N.

Heidbreder, M.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Heilemann, M.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Huang, B.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

Huang, F.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Huang, Z. L.

Jia, S.

Jones, S. A.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, Nat. Methods 8, 499 (2011).
[Crossref]

Joseph, N.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[Crossref]

Kanchanawong, P.

Z. Zhang, Y. Nishimura, and P. Kanchanawong, Mol. Biol. Cell 28, 333 (2017).
[Crossref]

Kechkar, A.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Kim, D.

Kirshner, H.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Klein, T.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Levet, F.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Li, P.

Lidke, K. A.

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

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[Crossref]

Lin, Y.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Long, F.

Long, J. J.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Löschberger, A.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Luo, Q.

Manley, S.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Min, J.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Mothes, W.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Myers, J. R.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Nienhaus, G. U.

Nieuwenhuizen, R. P.

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

Nishimura, Y.

Z. Zhang, Y. Nishimura, and P. Kanchanawong, Mol. Biol. Cell 28, 333 (2017).
[Crossref]

Nuwal, T.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

Owen, D. M.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Pengo, T.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Puig, D. L.

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

Quan, T.

Rieger, B.

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

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[Crossref]

Rivera-Molina, F. E.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Rossier, O.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Rubin-Delanchy, P.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Sage, D.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Sauer, M.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Schnitzbauer, J.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

Shim, S. H.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, Nat. Methods 8, 499 (2011).
[Crossref]

Sibarita, J. B.

A. Beghin, A. Kechkar, C. Butler, F. Levet, M. Cabillic, O. Rossier, G. Giannone, R. Galland, D. Choquet, and J. B. Sibarita, Nat. Methods 14, 1184 (2017).
[Crossref]

Sigal, Y. M.

Y. M. Sigal, R. Zhou, and X. Zhuang, Science 361, 880 (2018).
[Crossref]

Smith, C. S.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[Crossref]

Stallinga, S.

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

Stuurman, N.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Szymborska, A.

A. Szymborska, A. de Marco, N. Daigle, V. C. Cordes, J. A. Briggs, and J. Ellenberg, Science 341, 655 (2013).
[Crossref]

Toomre, D.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Uchil, P. D.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, Nat. Methods 10, 653 (2013).
[Crossref]

Unser, M.

D. Sage, H. Kirshner, T. Pengo, N. Stuurman, J. Min, S. Manley, and M. Unser, Nat. Methods 12, 717 (2015).
[Crossref]

Van de Linde, S.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Vaughan, J. C.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, Nat. Methods 8, 1027 (2011).
[Crossref]

Wang, Y.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
[Crossref]

Y. Wang, S. Jia, H. F. Zhang, D. Kim, H. Babcock, X. Zhuang, and L. Ying, Optica 4, 1277 (2017).
[Crossref]

Williamson, D. J.

P. Rubin-Delanchy, G. L. Burn, J. Griffié, D. J. Williamson, N. A. Heard, A. P. Cope, and D. M. Owen, Nat. Methods 12, 1072 (2015).
[Crossref]

Wolter, S.

S. Van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, Nat. Protoc. 6, 991 (2011).
[Crossref]

Ying, L.

Zeng, S.

Zhang, H. F.

Zhang, Z.

Z. Zhang, Y. Nishimura, and P. Kanchanawong, Mol. Biol. Cell 28, 333 (2017).
[Crossref]

Zhao, S.

J. Schnitzbauer, Y. Wang, S. Zhao, M. Bakalar, T. Nuwal, B. Chen, and B. Huang, Proc. Natl. Acad. Sci. USA 115, 3219 (2018).
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Proc. Natl. Acad. Sci. USA (1)

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Figures (4)

Fig. 1.
Fig. 1. Definition and calculation of SRI. (a) The relation between localizations and r-Spots. Previous (red cross) and new localizations (blue cross) are both Gaussian-distributed around their nearest r-Spot (black dot). (b) The normalized possibility density function of the distance between new localizations and their nearest r-Spot when σ = 10 nm . (c) Scheme of using SRI to end image acquisition.
Fig. 2.
Fig. 2. Relation between SRI and image acquisition time on simulated circle. (a), (d) Scattered images of molecules. (b), (e) Rendered images from the localizations obtained before SRI reaches 0.5. (c), (f) Time-dependent SRI (cyan squares) and rMole (blue dots). The red lines represent the SRI threshold (0.5). Data points are from 10 repeated measurements. Molecules are uniformly distributed in a circle with an interval distance of (a)–(c) 60 nm and (d)–(f) 12 nm, corresponding to visually nonoverlapping and overlapping molecule density, respectively. Localization precision, 10 nm. Scale bars, 150 nm.
Fig. 3.
Fig. 3. Image quality comparison with different ending criteria on simulated NPCs and microtubules. (a), (b) The relation among ending time, ending criteria, and localization precision. (a) and (b) are from NPCs and microtubules, respectively. Three criteria are compared here: FRC (red dots), SRI (green triangles), and rMole (blue squares). Data points are from 10 repeated measurements. (c)–(f) Rendered RGB (R, G, B channels are based on FRC, SRI, and rMole, respectively) and zoom-in single-channel images from accumulated localizations before each ending criterion is reached. Here (c), (d) NPCs and (e), (f) microtubules are simulated under different localization precision: 5 nm for (c), (e); 20 nm for (d), (f). Estimated complex radiuses of NPC across the full-field image are 41.3 nm (FRC), 44.0 nm (SRI), 44.3 nm (rMole) in (c), and 46.8 nm (FRC), 46.1 nm (SRI), 46.8 nm (rMole) in (d). (g), (h) Intensity profiles along the lines drawn in (e) and (f). FWHM in (g): 30.8 nm (FRC), 25.9 nm (SRI), 24.2 nm (rMole); FWHM in (h): 48.4 nm (FRC), 48.7 nm (SRI), 46.6 nm (rMole). Scale bars: 1 μm [(c)–(d), left panels], 90 nm [(c)–(d), right panels]; 2 μm [(e)–(f), top panels], 200 nm [(e)–(f), bottom panels].
Fig. 4.
Fig. 4. Image quality comparison with different ending criteria on experimental NPCs and microtubules. (a), (c) Rendered RGB (R, FRC; G, SRI; B, Whole) and zoom-in single-channel images from accumulated localizations before each ending criterion is reached [NPCs (a) and microtubules (c)]. Whole is rendered with localizations obtained from all raw images (20,000 frames for NPCs, and 10,000 frames for microtubules). (b), (d) Time-dependent SRI (green lines) and FRC (red dashes) values on NPCs (b) and microtubules (d). The arrows show the ending times based on SRI and FRC, respectively. (e) Intensity profiles along the line drawn in (c). FWHM: 66.9 nm (FRC), 68.2 nm (SRI), 65.3 nm (Whole). Scale bars: 3 μm [(a), left panel], 200 nm [(a), right panel]; 4 μm [(c), left panel], 500 nm [(c), right panel].

Equations (1)

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P ( D | σ ) = e D 2 / 2 σ 2 ,

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