Abstract

Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen’s inner structure.

© 2012 OSA

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References

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  1. M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc.198(2), 82–87 (2000).
    [CrossRef] [PubMed]
  2. R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” in Proc. SPIE. (SPIE, 1999), 3568, 185–196.
  3. L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
    [CrossRef] [PubMed]
  4. P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, “U.S. Patent 6016367,” (1997).
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  8. M. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  10. T. Kim, D. Gweon, and J. H. Lee, “Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination,” Meas. Sci. Technol.20(5), 055501 (2009).
    [CrossRef]
  11. R. Heintzmann, “Structured illumination methods,” in Handbook of Biological Confocal Microscopy (Springer, 2006), pp. 265–279.
  12. R. Heintzmann and P. A. Benedetti, “High-resolution image reconstruction in fluorescence microscopy with patterned excitation,” Appl. Opt.45(20), 5037–5045 (2006).
    [CrossRef] [PubMed]
  13. Kai. Wicker, PhD thesis, King’s College London, (2010).
  14. E. J. Botcherby, M. J. Booth, R. Juskaitis, and T. Wilson, “Real-time slit scanning microscopy in the meridional plane,” Opt. Lett.34(10), 1504–1506 (2009).
    [CrossRef] [PubMed]
  15. R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron34(6-7), 283–291 (2003).
    [CrossRef] [PubMed]

2012

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

2009

T. Kim, D. Gweon, and J. H. Lee, “Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination,” Meas. Sci. Technol.20(5), 055501 (2009).
[CrossRef]

E. J. Botcherby, M. J. Booth, R. Juskaitis, and T. Wilson, “Real-time slit scanning microscopy in the meridional plane,” Opt. Lett.34(10), 1504–1506 (2009).
[CrossRef] [PubMed]

2008

V. Poher, G. T. Kennedy, H. B. Manning, D. M. Owen, H. X. Zhang, E. Gu, M. D. Dawson, P. M. French, and M. A. Neil, “Improved sectioning in a slit scanning confocal microscope,” Opt. Lett.33(16), 1813–1815 (2008).
[CrossRef] [PubMed]

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

2006

2003

R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron34(6-7), 283–291 (2003).
[CrossRef] [PubMed]

2000

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

1997

1988

C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik (Stuttg.)80, 53–54 (1988).

1982

Agard, D. A.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Benedetti, P. A.

Booth, M. J.

Botcherby, E. J.

Burke, B.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Cardoso, M. C.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Carlton, P. M.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Chitnis, A. B.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Combs, C. A.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Cox, I. J.

Dawson, M. D.

Fischer, R. S.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

French, P. M.

Gu, E.

Gustafsson, M. G.

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

Gustafsson, M. G. L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Gweon, D.

T. Kim, D. Gweon, and J. H. Lee, “Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination,” Meas. Sci. Technol.20(5), 055501 (2009).
[CrossRef]

Haase, S.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Heintzmann, R.

R. Heintzmann and P. A. Benedetti, “High-resolution image reconstruction in fluorescence microscopy with patterned excitation,” Appl. Opt.45(20), 5037–5045 (2006).
[CrossRef] [PubMed]

R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron34(6-7), 283–291 (2003).
[CrossRef] [PubMed]

Juskaitis, R.

Kennedy, G. T.

Kim, T.

T. Kim, D. Gweon, and J. H. Lee, “Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination,” Meas. Sci. Technol.20(5), 055501 (2009).
[CrossRef]

Kner, P.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Lee, J. H.

T. Kim, D. Gweon, and J. H. Lee, “Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination,” Meas. Sci. Technol.20(5), 055501 (2009).
[CrossRef]

Leonhardt, H.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Manning, H. B.

Mione, M.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Neil, M. A.

Nogare, D. D.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Owen, D. M.

Parekh, S. H.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Poher, V.

Schermelleh, L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Sedat, J. W.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Shao, L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Sheppard, C. J. R.

Shroff, H.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Temprine, K.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Wilson, T.

Winoto, L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

York, A. G.

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Zhang, H. X.

Appl. Opt.

J. Microsc.

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

Meas. Sci. Technol.

T. Kim, D. Gweon, and J. H. Lee, “Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination,” Meas. Sci. Technol.20(5), 055501 (2009).
[CrossRef]

Micron

R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron34(6-7), 283–291 (2003).
[CrossRef] [PubMed]

Nat. Methods

A. G. York, S. H. Parekh, D. D. Nogare, 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. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Opt. Lett.

Optik (Stuttg.)

C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik (Stuttg.)80, 53–54 (1988).

Science

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science320(5881), 1332–1336 (2008).
[CrossRef] [PubMed]

Other

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, “U.S. Patent 6016367,” (1997).

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

R. Heintzmann, “Structured illumination methods,” in Handbook of Biological Confocal Microscopy (Springer, 2006), pp. 265–279.

Kai. Wicker, PhD thesis, King’s College London, (2010).

Supplementary Material (1)

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Figures (5)

Fig. 1
Fig. 1

Illustration of the LS-SIM setup. The black arrows indicate the movement of the line scan. Upper left inset: (a) one frame of the LS-SIM raw data In,o,p with indicated ON (illuminated) and OFF (not illuminated) regions (see further explanation in the main text). A close-up of the red box region is shown as (b) a wide-field SIM image I o,p WFSIM and (c) a line scan SIM I o,p LSSIM image. Scale bar (a-c) 2µm. Bottom right inset: Illustration of the LS-SIM pattern formation. Real space (top row) corresponds to a SIM grating plane. Fourier space (bottom row) represents the distribution of the intensity in the back focal plane (BFP) of the objective (h, i, j). The aperture of the BFP is indicated as a red circle. The multiplication (×) of the images in real space corresponds to the convolution operation () in Fourier space. The intensity distribution in the sample plane is shown in (g). The Fourier transform of (g) is shown in (k) with the border of the optical transfer function indicated as a green circle and the position of the Fourier transformed intensity orders of the grating are indicated with red arrows.

Fig. 2
Fig. 2

LS-SIM reconstructed image of a Calliphora salivary gland (i). WF image (bottom-left corner of (i)) shown for comparison. Scale bar 2 µm. Selected regions (red, green) reveal details of the actin structures: (a, b) WF image, (c, d) WF-SIM reconstruction, (e, f) LS image, (g, h) LS-SIM reconstruction. Intensity profiles along red line in (e) and blue line in (g) is plotted in (j) in corresponding colors. Scale bar (a-h) 1 µm.

Fig. 3
Fig. 3

Comparison of (a) LS-SIM and (b) a conventional WF-SIM reconstruction of the blue-framed region from Fig. 2(i). LS-SIM image is less affected with noise artifacts, which results in cleaner image. Arrows are pointing to the structures revealed in LS-SIM image. Scale bar 2µm.

Fig. 4
Fig. 4

Fourier transforms (a) of a single raw scan frame I n,o,p and (b) of a single I o,p LSSIM image computed from Eq. (2). Red arrows point at five peaks of the illumination pattern (see Fig. 1(k)). The second diffraction peaks are located at 70% of the cut-off frequency (green circle). The division of the diffraction lines into several points in (a) stems from the illumination of a multitude of lines per exposure.

Fig. 5
Fig. 5

Fourier transform of the LS image (a) with a green circle marking the cut-off frequency region. The Fourier transform of the reconstructed LS-SIM image (b) shows the extension of the transferred frequencies. The extended cut-off border is shown as a red circle. The arrows point at suspicious peaks in the spectrum possibly giving rise to the artifacts in the reconstructed image.

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

I o,p WFSIM = n=1 N I n,o,p .
I o,p LSSIM = max n ( I n,o,p )+ min n ( I n,o,p )2 mean n ( I n,o,p ),

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