Abstract

Photobleaching is a major factor limiting the observation time in fluorescence microscopy. We achieve photobleaching reduction in structured illumination microscopy (SIM) by locally adjusting the illumination intensities according to the sample. Adaptive SIM is enabled by a digital micro-mirror device (DMD), which provides a projection of the grayscale illumination patterns. We demonstrate a reduction in photobleaching by a factor of three in adaptive SIM compared to the non-adaptive SIM based on a spot grid scanning approach. Our proof-of-principle experiments show great potential for DMD-based microscopes to become a more useful tool in live-cell SIM imaging.

© 2016 Optical Society of America

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References

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  12. L. Shao, P. Kner, E. H. Rego, and M. G. L. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods 8, 1044–1046 (2011).
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  13. D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
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  14. S. Cox, “Super-resolution imaging in live cells,” Dev. Biol. 401, 175–181 (2015).
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    [Crossref]
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    [Crossref] [PubMed]
  24. K. K. Chu, D. Lim, and J. Mertz, “Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination,” Opt. Lett. 32, 2846–2848 (2007).
    [Crossref] [PubMed]
  25. T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19, 5644–5657 (2011).
    [Crossref] [PubMed]
  26. N. Chakrova, R. Heintzmann, B. Rieger, and S. Stallinga, “Studying different illumination patterns for resolution improvement in fluorescence microscopy,” Opt. Express 23, 31367–31383 (2015).
    [Crossref] [PubMed]
  27. L. Song, E. J. Hennik, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 66, 2588–2600 (1995).
    [Crossref]
  28. N. Chakrova, B. Rieger, and S. Stallinga, “Deconvolution methods for structured illumination microscopy,” J. Opt. Soc. Am. A 33, 12–20 (2016).
    [Crossref]
  29. N. Chakrova, B. Rieger, and S. Stallinga, “Development of a DMD-based fluorescence microscope,” Proc. SPIE 9330, 933008 (2015).
    [Crossref]
  30. J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216, 15–24 (2004).
    [Crossref] [PubMed]
  31. Z. Wang, A. C. Bovik, H. R. Sheikh, and E. P. Simoncelli, “Image quality assessment: From error visibility to structural similarity,” IEEE Trans. Sig. Process. 13, 600–612 (2004).
    [Crossref]

2016 (1)

N. Chakrova, B. Rieger, and S. Stallinga, “Deconvolution methods for structured illumination microscopy,” J. Opt. Soc. Am. A 33, 12–20 (2016).
[Crossref]

2015 (4)

N. Chakrova, B. Rieger, and S. Stallinga, “Development of a DMD-based fluorescence microscope,” Proc. SPIE 9330, 933008 (2015).
[Crossref]

N. Chakrova, R. Heintzmann, B. Rieger, and S. Stallinga, “Studying different illumination patterns for resolution improvement in fluorescence microscopy,” Opt. Express 23, 31367–31383 (2015).
[Crossref] [PubMed]

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

S. Cox, “Super-resolution imaging in live cells,” Dev. Biol. 401, 175–181 (2015).
[Crossref]

2013 (2)

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
[Crossref]

G. M. R. De Luca, R. M. P. Breedijk, R. Brandt, C. H. C. Zeelenberg, B. E. de Jong, W. Timmermans, L. N. Azar, R. Hoebe, S. Stallinga, and E. M. M. Manders, “Re-scan confocal microscopy: scanning twice for better resolution,” Biomed. Opt. Express 4, 2644–2656 (2013).
[Crossref] [PubMed]

2012 (3)

P. Křížek, I. Raška, and G. M. Hagen, “Flexible structured illumination microscope with a programmable illumination array,” Opt. Express 20, 24585–24599 (2012).
[Crossref]

E. Mudry, K. Belkebir, J. Girard, J. Savatier, E. Le Moal, C. Nicoletti, M. Allain, and A. Sentenac, “Structured illumination microscopy using unknown speckle patterns,” Nature Photon. 6, 312–315 (2012).
[Crossref]

A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
[Crossref] [PubMed]

2011 (3)

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope,” J. Microsc. 241, 101–110 (2011).
[Crossref]

L. Shao, P. Kner, E. H. Rego, and M. G. L. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods 8, 1044–1046 (2011).
[Crossref] [PubMed]

T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19, 5644–5657 (2011).
[Crossref] [PubMed]

2010 (1)

C. B. Muller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
[Crossref] [PubMed]

2009 (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, 339–342 (2009).
[Crossref] [PubMed]

2008 (1)

R. A. Hoebe, H. T. M. Van der Voort, J. Stap, C. J. F. Van Noorden, and E. M. M. Manders, “Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy,” J. Microsc. 231, 9–20 (2008).
[Crossref] [PubMed]

2007 (2)

K. K. Chu, D. Lim, and J. Mertz, “Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination,” Opt. Lett. 32, 2846–2848 (2007).
[Crossref] [PubMed]

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249–253 (2007).
[Crossref] [PubMed]

2006 (3)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, 793–795 (2006).
[Crossref] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258–4272 (2006).
[Crossref] [PubMed]

2004 (2)

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216, 15–24 (2004).
[Crossref] [PubMed]

Z. Wang, A. C. Bovik, H. R. Sheikh, and E. P. Simoncelli, “Image quality assessment: From error visibility to structural similarity,” IEEE Trans. Sig. Process. 13, 600–612 (2004).
[Crossref]

2000 (3)

G. E. Cragg and P. T. So, “Lateral resolution enhancement with standing evanescent waves,” Opt. Lett. 25, 46–48 (2000).
[Crossref]

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

J. T. Frohn, H. F. Knapp, and A. Stemmer, “True optical resolution beyond the Rayleigh limit achieved by standing wave illumination,” Proc. Natl. Acad. Sci. USA 93, 7232–7236 (2000).
[Crossref]

1999 (2)

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

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, and T. M. Jovin, “An optical sectioning programmable array microscope implemented with a digital micromirror device,” J. Microsc. 196, 317–331 (1999).
[Crossref] [PubMed]

1998 (1)

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. v. Vliet, and T. M. Jovin, “Theory of confocal fluorescence imaging in the programmable array microscope (PAM),” J. Microsc. 189, 192–198 (1998).
[Crossref]

1995 (1)

L. Song, E. J. Hennik, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 66, 2588–2600 (1995).
[Crossref]

1994 (1)

Allain, M.

E. Mudry, K. Belkebir, J. Girard, J. Savatier, E. Le Moal, C. Nicoletti, M. Allain, and A. Sentenac, “Structured illumination microscopy using unknown speckle patterns,” Nature Photon. 6, 312–315 (2012).
[Crossref]

Arndt-Jovin, D. J.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope,” J. Microsc. 241, 101–110 (2011).
[Crossref]

Azar, L. N.

Baird, M. A.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, 793–795 (2006).
[Crossref] [PubMed]

Beach, J. R.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

Belkebir, K.

E. Mudry, K. Belkebir, J. Girard, J. Savatier, E. Le Moal, C. Nicoletti, M. Allain, and A. Sentenac, “Structured illumination microscopy using unknown speckle patterns,” Nature Photon. 6, 312–315 (2012).
[Crossref]

Betzig, E.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Bovik, A. C.

Z. Wang, A. C. Bovik, H. R. Sheikh, and E. P. Simoncelli, “Image quality assessment: From error visibility to structural similarity,” IEEE Trans. Sig. Process. 13, 600–612 (2004).
[Crossref]

Brakenhoff, G. J.

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216, 15–24 (2004).
[Crossref] [PubMed]

Brandt, R.

Breedijk, R. M. P.

Caarls, W.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope,” J. Microsc. 241, 101–110 (2011).
[Crossref]

Chakrova, N.

N. Chakrova, B. Rieger, and S. Stallinga, “Deconvolution methods for structured illumination microscopy,” J. Opt. Soc. Am. A 33, 12–20 (2016).
[Crossref]

N. Chakrova, B. Rieger, and S. Stallinga, “Development of a DMD-based fluorescence microscope,” Proc. SPIE 9330, 933008 (2015).
[Crossref]

N. Chakrova, R. Heintzmann, B. Rieger, and S. Stallinga, “Studying different illumination patterns for resolution improvement in fluorescence microscopy,” Opt. Express 23, 31367–31383 (2015).
[Crossref] [PubMed]

Chen, B.-C.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

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, 339–342 (2009).
[Crossref] [PubMed]

Chitnis, A. B.

A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
[Crossref] [PubMed]

Chu, K. K.

Combs, C. a.

A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
[Crossref] [PubMed]

Cox, S.

S. Cox, “Super-resolution imaging in live cells,” Dev. Biol. 401, 175–181 (2015).
[Crossref]

Cragg, G. E.

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]

DalleNogare, D.

A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
[Crossref] [PubMed]

Dan, D.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
[Crossref]

Davidson, M. W.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

de Jong, B. E.

De Luca, G. M. R.

De Vries, A. H. B.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope,” J. Microsc. 241, 101–110 (2011).
[Crossref]

Dhonukshe, P. B.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249–253 (2007).
[Crossref] [PubMed]

Enderlein, J.

C. B. Muller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
[Crossref] [PubMed]

Engelhardt, J.

Engler, A.

Fischer, R. S.

A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
[Crossref] [PubMed]

Frohn, J. T.

J. T. Frohn, H. F. Knapp, and A. Stemmer, “True optical resolution beyond the Rayleigh limit achieved by standing wave illumination,” Proc. Natl. Acad. Sci. USA 93, 7232–7236 (2000).
[Crossref]

Gadella, T. W. J.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249–253 (2007).
[Crossref] [PubMed]

Gao, P.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
[Crossref]

Gemkow, M. J.

Q. S. Hanley, P. J. Verveer, M. J. Gemkow, and T. M. Jovin, “An optical sectioning programmable array microscope implemented with a digital micromirror device,” J. Microsc. 196, 317–331 (1999).
[Crossref] [PubMed]

Girard, J.

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S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258–4272 (2006).
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J. T. Frohn, H. F. Knapp, and A. Stemmer, “True optical resolution beyond the Rayleigh limit achieved by standing wave illumination,” Proc. Natl. Acad. Sci. USA 93, 7232–7236 (2000).
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L. Shao, P. Kner, E. H. Rego, and M. G. L. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods 8, 1044–1046 (2011).
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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, 339–342 (2009).
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Le Moal, E.

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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
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E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
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S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258–4272 (2006).
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D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
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D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
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E. Mudry, K. Belkebir, J. Girard, J. Savatier, E. Le Moal, C. Nicoletti, M. Allain, and A. Sentenac, “Structured illumination microscopy using unknown speckle patterns,” Nature Photon. 6, 312–315 (2012).
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D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
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L. Shao, P. Kner, E. H. Rego, and M. G. L. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods 8, 1044–1046 (2011).
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E. Mudry, K. Belkebir, J. Girard, J. Savatier, E. Le Moal, C. Nicoletti, M. Allain, and A. Sentenac, “Structured illumination microscopy using unknown speckle patterns,” Nature Photon. 6, 312–315 (2012).
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Stap, J.

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L. Song, E. J. Hennik, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 66, 2588–2600 (1995).
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A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
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R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249–253 (2007).
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J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216, 15–24 (2004).
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Q. S. Hanley, P. J. Verveer, M. J. Gemkow, and T. M. Jovin, “An optical sectioning programmable array microscope implemented with a digital micromirror device,” J. Microsc. 196, 317–331 (1999).
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P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. v. Vliet, and T. M. Jovin, “Theory of confocal fluorescence imaging in the programmable array microscope (PAM),” J. Microsc. 189, 192–198 (1998).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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Z. Wang, A. C. Bovik, H. R. Sheikh, and E. P. Simoncelli, “Image quality assessment: From error visibility to structural similarity,” IEEE Trans. Sig. Process. 13, 600–612 (2004).
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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, 339–342 (2009).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination Super-resolution and optical sectioning microscopy,” Sci. Rep. 3, 01116 (2013).
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A. G. York, S. H. Parekh, D. DalleNogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. a. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9, 749–754 (2012).
[Crossref] [PubMed]

Young, I. T.

L. Song, E. J. Hennik, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 66, 2588–2600 (1995).
[Crossref]

Zeelenberg, C. H. C.

Zhang, M.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

Zhang, X.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349, aab3500 (2015).
[Crossref]

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Figures (7)

Fig. 1
Fig. 1

Block diagram of time-lapse adaptive SIM.

Fig. 2
Fig. 2

Two approaches for grayscale mask generation. In mask (a) the weight of the grayscale mask depends linearly on the intensities in the widefield image. In mask (b) the weight of the grayscale mask is inversely proportional to the intensities in the widefield image. Boundary values Imin and Imax are found empirically for a given sample.

Fig. 3
Fig. 3

Photobleaching curves were acquired in a widefield mode at different illumination intensities on a sample containing HEK 293T cells in which the DNA was labeled with SYTOX green nucleic acid stain. In the region of illumination intensities 0 – 10 W/cm2 photobleaching rate depends linearly on the illumination intensity. Additionally, a small self-quenching effect is observable at initial times due to the dense DNA labeling.

Fig. 4
Fig. 4

Widefield image of HEK 293T cells in which DNA is labelled with SYTOX stain (a), and the resulting grayscale masks (b–d), which can be used as a weight for the multi-spot illumination patterns.

Fig. 5
Fig. 5

Comparison of standard and adaptive SIM imaging modalities. (a) Visual similarity of the adaptive and standard SIM images acquired under equal imaging conditions. Since the peak intensity of the illumination in adaptive and non-adaptive SIM is the same, adaptive SIM reconstruction has a similar range of intensities as the standard SIM reconstruction. (b) The sum of 100 raw images in standard SIM results in a widefield image, whereas the sum of 100 raw images in adaptive SIM results in an image with a more unified signal level. (c) Examples of the illumination patterns for the standard and adaptive SIM. Illumination patterns for the adaptive SIM are produced by multiplying the standard SIM illumination patterns with the mask shown in Fig. 4(c).

Fig. 6
Fig. 6

Comparison of photobleaching in standard and adaptive SIM. Each curve shows the average of 5 measurements on separate sample areas and the error bars indicate the standard deviations over 5 measurements. Adaptive SIM enables at least three times longer imaging than the standard SIM.

Fig. 7
Fig. 7

Comparison of the photobleaching induced by 30 minutes of time-lapse imaging in standard and adaptive SIM on HEK 293T cells, in which DNA is labelled with SYTOX stain. (a) First acquisition, (b) 20th acquisition (10 min), (c) 40th acquisition (20 min), (d) 60th acquisition (30 min). Adaptive SIM leads to deceleration of the photobleaching, enabling longer observation time of the sample. Intensities are comparable over all images.

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