Abstract

The reconstruction process of structured illumination microscopy (SIM) creates substantial artefacts if the specimen has moved during the acquisition. This reduces the applicability of SIM for live cell imaging, because these artefacts cannot always be recognized as such in the final image. A movement is not necessarily visible in the raw data, due to the varying excitation patterns and the photon noise. We present a method to detect motion by extracting and comparing two independent 3D wide-field images out of the standard SIM raw data without needing additional images. Their difference reveals moving objects overlaid with noise, which are distinguished by a probability theory-based analysis. Our algorithm tags motion-artefacts in the final high-resolution image for the first time, preventing the end-user from misinterpreting the data. We show and explain different types of artefacts and demonstrate our algorithm on a living cell.

Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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References

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  1. R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185–196 (1999).
    [Crossref]
  2. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
    [Crossref] [PubMed]
  3. M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
    [Crossref] [PubMed]
  4. Nikon Corporation, “Super resolution microscope N-SIM E,” https://www.nikoninstruments.com/content/download/18277/440857/file/N-SIM_E_2CE-SCMH.pdf .
  5. G. E. Healthcare, “DeltaVisionTM OMX SR super-resolution microscope,” https://www.gelifesciences.com/gehcls_images/GELS/Related Content/Files/1435762098806/litdoc29146719_20150701164816.pdf .
  6. L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
    [Crossref] [PubMed]
  7. P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
    [Crossref] [PubMed]
  8. R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
    [Crossref] [PubMed]
  9. H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
    [Crossref]
  10. L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
    [Crossref]
  11. T. Xia, N. Li, and X. Fang, “Single-molecule fluorescence imaging in living cells,” Annu. Rev. Phys. Chem. 64(1), 459–480 (2013).
    [Crossref] [PubMed]
  12. K. Wicker and R. Heintzmann, “Single-shot optical sectioning using polarization-coded structured illumination,” J. Opt. 12(8), 084010 (2010).
    [Crossref]
  13. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer Science, 2006).
  14. I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
    [Crossref] [PubMed]

2016 (1)

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

2015 (1)

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

2014 (1)

2013 (1)

T. Xia, N. Li, and X. Fang, “Single-molecule fluorescence imaging in living cells,” Annu. Rev. Phys. Chem. 64(1), 459–480 (2013).
[Crossref] [PubMed]

2012 (1)

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

2010 (1)

K. Wicker and R. Heintzmann, “Single-shot optical sectioning using polarization-coded structured illumination,” J. Opt. 12(8), 084010 (2010).
[Crossref]

2009 (2)

L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
[Crossref] [PubMed]

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

2008 (1)

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

2000 (1)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

1999 (1)

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185–196 (1999).
[Crossref]

Agard, D. A.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Cande, W. Z.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Carlton, P. M.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Chhun, B. B.

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

Cremer, C.

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185–196 (1999).
[Crossref]

Fang, X.

T. Xia, N. Li, and X. Fang, “Single-molecule fluorescence imaging in living cells,” Annu. Rev. Phys. Chem. 64(1), 459–480 (2013).
[Crossref] [PubMed]

Förster, R.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
[Crossref] [PubMed]

Garstka, M.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Gieczewska, K.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Golubovskaya, I. N.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Griffis, E. R.

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

Gustafsson, M. G. L.

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

Heintzmann, R.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
[Crossref] [PubMed]

K. Wicker and R. Heintzmann, “Single-shot optical sectioning using polarization-coded structured illumination,” J. Opt. 12(8), 084010 (2010).
[Crossref]

L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
[Crossref] [PubMed]

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185–196 (1999).
[Crossref]

Hirvonen, L. M.

L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
[Crossref] [PubMed]

Jost, A.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
[Crossref] [PubMed]

Kielhorn, M.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
[Crossref] [PubMed]

Kierdaszuk, B.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Kner, P.

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

Koziol-Lipinska, J.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Li, N.

T. Xia, N. Li, and X. Fang, “Single-molecule fluorescence imaging in living cells,” Annu. Rev. Phys. Chem. 64(1), 459–480 (2013).
[Crossref] [PubMed]

Lu-Walther, H.-W.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
[Crossref] [PubMed]

Mandula, O.

L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
[Crossref] [PubMed]

Mazur, R.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Michalski, W. P.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Mostowska, A.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Rumak, I.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Sedat, J. W.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Shao, L.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Shiell, B. J.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Song, L.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

Venema, J. H.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Vredenberg, W. J.

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Wang, C. J. R.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

Wicker, K.

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663–20677 (2014).
[Crossref] [PubMed]

K. Wicker and R. Heintzmann, “Single-shot optical sectioning using polarization-coded structured illumination,” J. Opt. 12(8), 084010 (2010).
[Crossref]

L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
[Crossref] [PubMed]

Winoto, L.

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

Xia, T.

T. Xia, N. Li, and X. Fang, “Single-molecule fluorescence imaging in living cells,” Annu. Rev. Phys. Chem. 64(1), 459–480 (2013).
[Crossref] [PubMed]

Zhou, J.

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

Annu. Rev. Phys. Chem. (1)

T. Xia, N. Li, and X. Fang, “Single-molecule fluorescence imaging in living cells,” Annu. Rev. Phys. Chem. 64(1), 459–480 (2013).
[Crossref] [PubMed]

Biophys. J. (1)

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008).
[Crossref] [PubMed]

BMC Plant Biol. (1)

I. Rumak, R. Mazur, K. Gieczewska, J. Kozioł-Lipińska, B. Kierdaszuk, W. P. Michalski, B. J. Shiell, J. H. Venema, W. J. Vredenberg, A. Mostowska, and M. Garstka, “Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes,” BMC Plant Biol. 12(1), 72 (2012).
[Crossref] [PubMed]

Eur. Biophys. J. (1)

L. M. Hirvonen, K. Wicker, O. Mandula, and R. Heintzmann, “Structured illumination microscopy of a living cell,” Eur. Biophys. J. 38(6), 807–812 (2009).
[Crossref] [PubMed]

J. Microsc. (1)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

J. Opt. (1)

K. Wicker and R. Heintzmann, “Single-shot optical sectioning using polarization-coded structured illumination,” J. Opt. 12(8), 084010 (2010).
[Crossref]

Meas. Sci. Technol. (1)

L. Song, H.-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann, “Fast structured illumination microscopy using rolling shutter cameras,” Meas. Sci. Technol. 27(5), 055401 (2016).
[Crossref]

Methods Appl. Fluoresc. (1)

H.-W. Lu-Walther, M. Kielhorn, R. Förster, A. Jost, K. Wicker, and R. Heintzmann, “fastSIM: a practical implementation of fast structured illumination microscopy,” Methods Appl. Fluoresc. 3(1), 014001 (2015).
[Crossref]

Nat. Methods (1)

P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6(5), 339–342 (2009).
[Crossref] [PubMed]

Opt. Express (1)

Proc. SPIE (1)

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185–196 (1999).
[Crossref]

Other (3)

Nikon Corporation, “Super resolution microscope N-SIM E,” https://www.nikoninstruments.com/content/download/18277/440857/file/N-SIM_E_2CE-SCMH.pdf .

G. E. Healthcare, “DeltaVisionTM OMX SR super-resolution microscope,” https://www.gelifesciences.com/gehcls_images/GELS/Related Content/Files/1435762098806/litdoc29146719_20150701164816.pdf .

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer Science, 2006).

Supplementary Material (3)

NameDescription
» Visualization 1: AVI (1827 KB)      3D SIM stack of a moving chloroplast
» Visualization 2: AVI (7425 KB)      3D stack of motion detection processing steps of a floating chloroplast
» Visualization 3: AVI (9869 KB)      3D stack of motion detection processing steps of a fixed stoma

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Figures (10)

Fig. 1
Fig. 1

Summary three-beam SIM: The specimen S is illuminated with several fine sinusoidal excitation patterns Iφ (top row). High sample frequencies are down-modulated and form coarse interference fringes (middle row), which are well transferred by the objective. The images Dφ (bottom row) are formed by a convolution with the PSF h. The reconstruction algorithm converts the down-modulated low-frequency fringes back to their original frequency, which is beyond the Abbe-limit. Thus the SIM image has a higher resolution than the wide-field image D. The necessary conversion needs typically ϕ = 5 different raw images, to separate 5 different intensity orders overlapping in Fourier space. Thus, 5 images Dφ of the sample are acquired with 5 different excitation patterns Iφ, which differ only in a lateral shift called phase φ. The small shift can be seen best at the fixed yellow reference line (for visualization purpose only). All five excitation patterns add up to a homogeneous pattern Isum, so that no residual pattern is bleached into the specimen. In order to achieve an isotropic resolution enhancement, this procedure has to be repeated three times, while the grating is rotated by 60° after every five images. Hence there are 15 raw images per focus slice. Acquiring them at every focus position creates a 3D raw data stack, which contains typically around 300 million pixels ([x,y,ϕ,z] = [1000,1000,15,20]) for each colour and timeframe.

Fig. 2
Fig. 2

Overview of SIM orders in Fourier space: a) 3 excitation beams E ˜ φ ( k ) with phase φ are focused in the back focal plane (BFP) of the objective (orange dots). They form three interfering plane waves E φ ( r ) in the specimen. b) Complex conjugated beams E φ * ~ ( k ) ; c) The excitation pattern in the specimen I φ ( r ) is E ˜ φ ( k ) E φ * ~ ( k ) . As a result, the excitation pattern represented in Fourier space is the autocorrelation of the incoming beams: I ˜ φ ( k )= E ˜ φ ( k ) E φ * ~ ( k ) . This leads to 7 SIM illumination intensity peaks (orange dots). d) The sample information is attached to each of them, causing the down modulation of high frequency information, before the multiplicative low-pass of the OTF (shown in e) damps or removes it. This artificial frequency shift needs to be undone so that the precious transferred high frequency components are placed at their true original position. However, all SIM orders are always present at the same time in each single raw image, so that they need to be separated from each other. This can be done by exploiting the individual phase of each illumination intensity peak, which can be controlled by a shift of the excitation pattern in the sample plane. Thus, SIM needs as many images with different phases as orders. Unfortunately, the first order pairs modulate identical and cannot be separated. However, they only differ by their axial shift in Fourier space (blue line in panel d). Since the 3D illumination structure is refocussing with the sample refocus, each such pair automatically demodulates, leading to an axially enlarged effective detection OTF (blue OTF in panel g). Thus, only 5 independent SIM orders remain (panel f) with different effective detection OTFs. However, the axial sampling has to be doubled to avoid aliasing in the axial direction because of the extended frequency support of the two detection OTFs [1–3].

Fig. 3
Fig. 3

Adding up all phases gives a standard wide-field image which is axially oversampled by a factor of two. Two independent wide-field image z-stacks are generated by separating even and odd slices. Their different focus position z is corrected in the Fourier domain. The frame-difference method can be applied to them.

Fig. 4
Fig. 4

Hypothesis test if the two measured values D even ( IL ) ( r ) and D odd ( IL ) ( r ) originate from the same, but unknown, Gaussian probability distribution (top left). Thus, the difference of both measurementsΔis scaled to its estimated standard deviation σ[ Δ( r ) ] , which is the z-score z. (top right). If z is bigger than 6.5, the hypothesis is rejected and a movement must have occurred in the corresponding pixel (bottom right).

Fig. 5
Fig. 5

Wide-field images out of a SIM z-stack focussed on a) a free chloroplast and b) a more stable stoma within the epidermis. Both layers are 5 µm apart.

Fig. 6
Fig. 6

Wide-field z-stack of a moving chloroplast out of SIM raw data (γ = 1,8). a) z = z0 and c) z = z0 + 140nm. The thylakoid moves out of the red box, which cannot be explained by the axial shape of the PSF. Thus, it has to be motion. b) The movement of each thylakoid is tracked by hand and displayed by the rods. The rotation axis is marked blue.

Fig. 7
Fig. 7

a) wide-field and b) SIM image of a moving chloroplast. The SIM image provides higher resolution in a slice-per-slice reconstruction. However, the three-dimensional shape is distorted (Visualization 1).

Fig. 8
Fig. 8

a) wide-field and b) SIM image of a fast moving fluorophore. The reconstruction leads to a strong artefact.

Fig. 9
Fig. 9

Image Processing Steps marked by the yellow arrows (γ = 0.35): a) D odd ( IL ) ( r ) with motion in red; b) σ[ Δ( r ) ] ; c) z( r )>6.5 ; d) border of Z h ( r ) ; e)  | Δ( r ) | ; f) z( r ) ; g) Z h ( r ) ; h) SIM-image with motion in red (Visualization 2 and Visualization 3).

Fig. 10
Fig. 10

SIM image of the stoma (left) and moving chloroplast (right). Movement artefacts are detected and encircled in red. (γ = 0.25 for a better visibility of low light artefacts.)

Equations (14)

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D( r )= φ=1 5 D φ ( r )= φ=1 5 ( ( S I φ )h )( r )=( ( S φ=1 5 I φ )h )( r ) =( ( S I sum )h )( r )  ( Sh )( r )
K ˜ up/down ( k z )=  e ± iπ k z 2  k sampling
D even ( IL ) ( r )=( D even K up )( r )
D odd ( IL ) ( r )=( D odd K down )( r )
Δ( r )=  D even ( IL ) ( r ) D odd ( IL ) ( r )~ ( S ( IL ) ( r , t 0 ) S ( IL ) ( r , t 0 +Δt ) )h motion + Poisson and readout noise
σ pois 2 [ g D φ ( r ) ]= g 2 σ pois 2 [ D φ ( r ) ] σ pois 2 [ g D φ ( r ) ]= μ[ g D φ ( r ) ]g D φ ( r )+1 σ pois 2 [ D φ ( r ) ]  D φ ( r ) g + 1 g 2
σ 2 [ D φ ( r ) ]=  σ pois 2 [ D φ ( r ) ]+ σ dark 2
σ 2 [ D( r ) ]=  φ σ 2 [ D φ ( r ) ]
σ 2 [ D even ( IL ) ( r ) ]=( σ 2 [ D even ( r ) ]  K up 2 ( z ) )( r )
σ 2 [ D odd ( IL ) ( r ) ]=( σ 2 [ D odd ( r ) ]   K odd 2 ( z ) )( r )
σ 2 [ Δ( r ) ]= σ 2 [ D even ( IL ) ( r ) ] +  σ 2 [ D odd ( IL ) ( r ) ]
z( r ):= | Δ( r )μ[ Δ( r ) ] | σ[ Δ( r ) ] = | Δ( r ) | σ[ Δ( r ) ]
P G ( r )=P( zz( r ) | G( 0,σ[ Δ( r ) ] ) ) =1P( z<z( r ) | G( 0,σ[ Δ( r ) ] ) )=1erf( z( r ) 2 )
z h ( r )= | ( Δh )( r ) | ( σ 2 ( h 2 ) )( r )

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