Abstract

The use of structured illumination in fluorescence microscopy allows the suppression of out of focus light and an increase in effective spatial resolution. In this paper we consider different approaches for reconstructing 2D structured illumination images in order to combine these two attributes, to allow fast, optically sectioned, superresolution imaging. We present a linear reconstruction method that maximizes the axial frequency extent of the combined 2D structured illumination passband along with an empirically optimized approximation to this scheme. These reconstruction methods are compared to other schemes using structured illumination images of fluorescent samples. For sinusoidal excitation at half the incoherent cutoff frequency we find that removing information in the zero order passband except for a small region close to the excitation frequency, where it replaces the complementary information from the displaced first order passband, enables optimal reconstruction of optically sectioned images with enhanced spatial resolution.

© 2014 Optical Society of America

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References

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  1. R. Heintzmann, “Structured Illumination Methods,” in Handbook Of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer US, 2006), pp. 265–279.
  2. M. A. 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]
  3. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc.198, 82–87 (2000).
  4. R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE3568, 185–196 (1999).
    [CrossRef]
  5. B. Thomas, M. Momany, and P. Kner, “Optical sectioning structured illumination microscopy with enhanced sensitivity,” J. Opt.15(9), 094004 (2013).
    [CrossRef]
  6. K. Wicker, O. Mandula, G. Best, R. Fiolka, and R. Heintzmann, “Phase optimisation for structured illumination microscopy,” Opt. Express21(2), 2032–2049 (2013).
    [CrossRef] [PubMed]
  7. 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]
  8. D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
    [CrossRef] [PubMed]
  9. J. Philip, “Optical transfer function in three dimensions for a large numerical aperture,” J. Mod. Opt.46(6), 1031–1042 (1999).
    [CrossRef]
  10. 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. Methods6(5), 339–342 (2009).
    [CrossRef] [PubMed]
  11. K. O’Holleran and M. Shaw, “Polarization effects on contrast in structured illumination microscopy,” Opt. Lett.37(22), 4603–4605 (2012).
    [CrossRef] [PubMed]
  12. T. Wilson, “Resolution and optical sectioning in the confocal microscope,” J. Microsc.244(2), 113–121 (2011).
    [CrossRef] [PubMed]
  13. M. G. Somekh, K. Hsu, and M. C. Pitter, “Stochastic transfer function for structured illumination microscopy,” J. Opt. Soc. Am. A26(7), 1630–1637 (2009).
    [CrossRef] [PubMed]

2013

B. Thomas, M. Momany, and P. Kner, “Optical sectioning structured illumination microscopy with enhanced sensitivity,” J. Opt.15(9), 094004 (2013).
[CrossRef]

K. Wicker, O. Mandula, G. Best, R. Fiolka, and R. Heintzmann, “Phase optimisation for structured illumination microscopy,” Opt. Express21(2), 2032–2049 (2013).
[CrossRef] [PubMed]

2012

2011

T. Wilson, “Resolution and optical sectioning in the confocal microscope,” J. Microsc.244(2), 113–121 (2011).
[CrossRef] [PubMed]

2009

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. Methods6(5), 339–342 (2009).
[CrossRef] [PubMed]

M. G. Somekh, K. Hsu, and M. C. Pitter, “Stochastic transfer function for structured illumination microscopy,” J. Opt. Soc. Am. A26(7), 1630–1637 (2009).
[CrossRef] [PubMed]

2008

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]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

2000

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

1999

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE3568, 185–196 (1999).
[CrossRef]

J. Philip, “Optical transfer function in three dimensions for a large numerical aperture,” J. Mod. Opt.46(6), 1031–1042 (1999).
[CrossRef]

1997

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]

Best, G.

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. Methods6(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. SPIE3568, 185–196 (1999).
[CrossRef]

Fiolka, R.

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. Methods6(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. Methods6(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, 82–87 (2000).

Heintzmann, R.

K. Wicker, O. Mandula, G. Best, R. Fiolka, and R. Heintzmann, “Phase optimisation for structured illumination microscopy,” Opt. Express21(2), 2032–2049 (2013).
[CrossRef] [PubMed]

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE3568, 185–196 (1999).
[CrossRef]

Hsu, K.

Juskaitis, R.

Karadaglic, D.

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

Kner, P.

B. Thomas, M. Momany, and P. Kner, “Optical sectioning structured illumination microscopy with enhanced sensitivity,” J. Opt.15(9), 094004 (2013).
[CrossRef]

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. Methods6(5), 339–342 (2009).
[CrossRef] [PubMed]

Mandula, O.

Momany, M.

B. Thomas, M. Momany, and P. Kner, “Optical sectioning structured illumination microscopy with enhanced sensitivity,” J. Opt.15(9), 094004 (2013).
[CrossRef]

Neil, M. A. A.

O’Holleran, K.

Philip, J.

J. Philip, “Optical transfer function in three dimensions for a large numerical aperture,” J. Mod. Opt.46(6), 1031–1042 (1999).
[CrossRef]

Pitter, M. C.

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]

Shaw, M.

Somekh, M. G.

Thomas, B.

B. Thomas, M. Momany, and P. Kner, “Optical sectioning structured illumination microscopy with enhanced sensitivity,” J. Opt.15(9), 094004 (2013).
[CrossRef]

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.

Wilson, T.

T. Wilson, “Resolution and optical sectioning in the confocal microscope,” J. Microsc.244(2), 113–121 (2011).
[CrossRef] [PubMed]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

M. A. 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]

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. Methods6(5), 339–342 (2009).
[CrossRef] [PubMed]

Biophys. J.

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]

J. Microsc.

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

T. Wilson, “Resolution and optical sectioning in the confocal microscope,” J. Microsc.244(2), 113–121 (2011).
[CrossRef] [PubMed]

J. Mod. Opt.

J. Philip, “Optical transfer function in three dimensions for a large numerical aperture,” J. Mod. Opt.46(6), 1031–1042 (1999).
[CrossRef]

J. Opt.

B. Thomas, M. Momany, and P. Kner, “Optical sectioning structured illumination microscopy with enhanced sensitivity,” J. Opt.15(9), 094004 (2013).
[CrossRef]

J. Opt. Soc. Am. A

Micron

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

Nat. Methods

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. Methods6(5), 339–342 (2009).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Proc. SPIE

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE3568, 185–196 (1999).
[CrossRef]

Other

R. Heintzmann, “Structured Illumination Methods,” in Handbook Of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer US, 2006), pp. 265–279.

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Figures (5)

Fig. 1
Fig. 1

(a) Axial (Z) extent of 3D OTF support in the ( k x , k z ) plane for D 0,1 (red) and D ±1,1 (green). (b) 1D illustration showing the frequency range over which information is included from the zero (red) and first order (green) passbands in the Max kz reconstruction scheme. (c) Weighted combination of information from zero (red) and first order (green) passbands in the WLR method. (d) Boundary of OTF support with 3 orientations of ω when | ω | is equal to half the incoherent cutoff frequency. (e) Combination of the zero and first order passbands which maximises the axial support of the OTF for all spatial frequencies. (f) Combination of the zero and first order passbands in the Max kz method. (g) Weighted combination of zero and first order passbands in the WLR method. For clarity, only the weighting for each D 0,n (red) and a single weighting for D +1,1 (green) are shown.

Fig. 2
Fig. 2

Schematic diagram of the SIM system used to acquire images for testing reconstruction algorithms. Lenses L1-L4 are achromatic doublets, SLM is a ferroelectric liquid crystal on silicon spatial light modulator, M is a plane mirror and MB is the microscope body. Inset I1 shows regions of the phase gratings displayed on SLM for 3 different excitation pattern orientations. Inset III shows the mask in the Fourier plane of the SLM used to select the positive and negative first diffracted orders. Inset IIII shows a sinusoidal intensity pattern at the focal plane of the microscope objective lens (MO).

Fig. 3
Fig. 3

(a) Widefield and SI images of 0.17 μm diameter yellow-green fluorescent microspheres dried on a glass coverslip. Scale bar is 1 μm. (b) Line profiles drawn through a single isolated microsphere. Measured FWHM is 0.30 μm, 0.21 μm, 0.27 μm, 0.17 μm, 0.18 μm, 0.17 μm for widefield, SR, OS, LROS, Max kz and WLR reconstructions respectively. (c) Line profiles drawn through two fluorescent microspheres in a part of the image containing a densely packed monolayer of microspheres.

Fig. 4
Fig. 4

(a) Axial response from images of a single yellow-green fluorescent microsphere reconstructed using different SI algorithms. (b) FWHM of axial response versus lateral FWHM of each method averaged over 10 individual fluorescent microspheres. Horizontal and vertical axes of ellipses show ±1 standard deviation in the measured FWHMs. Mean measured lateral and axial FWHM values, in µm, are (0.28, 0.70), (0.20, 0.74), (0.27, 0.66), (0.17, 0.64), (0.18, 0.54) and (0.17, 0.59) for the widefield, SR, OS, LROS, Max kz and WLR reconstructions respectively.

Fig. 5
Fig. 5

(a) Images of microtubules in a HeLa cell. Staining for tubulin with rat anti-tubulin primary and anti-rat-Alexa 488 secondary antibodies. (b) Magnified images of boxed regions in main images. FWHM of the axial response for a small region close to the center of the image was 0.82 μm, 0.71 μm, 0.67 μm and 0.66 μm for OS, LROS, Max kz and WLR methods respectively. (c) Projections of a 3D rendering of the z-stack of images formed using the WLR reconstruction. Scale bars are 4 μm. (d) Line profiles along the coloured lines shown in (b).

Equations (4)

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1 2 [ 2 2 2 e i ϕ 1 e i ϕ 2 e i ϕ 3 e i ϕ 1 e i ϕ 2 e i ϕ 3 ][ D(k) D(k+ω) D(kω) ]=[ E(k, ϕ 1 ) E(k, ϕ 2 ) E(k, ϕ 3 ) ],
I ˜ max k z =(1(k))( n=1 N 1 N D 0,n (k) )+(k)( n=1 N D +1,n (k)+ D 1,n (k) )
a( k,ω )=exp[ ( k,ω ) 2 /2 k σ 2 ],
I WLR (k)= n=1 N [a( k, ω n )+a( k, ω n )] 1 N D 0,n (k)+ [1a( k, ω n )] D +1,n (k)+[1a( k, ω n )] D 1,n (k) ,

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