Abstract

The effect of detector array size on resolution and signal collection efficiency of image scanning microscopy based on pixel reassignment is studied. It is shown how the method can also be employed if there is a Stokes shift in fluorescence emission wavelength. With no Stokes shift, the width of the point spread function can be sharpened by a factor of 1.53, and its peak intensity increased by a factor of 1.84.

© 2013 Optical Society of America

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References

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  1. C. B. Müller and J. Enderlein, Phys. Rev. Lett. 104, 198101 (2010).
    [CrossRef]
  2. H. Blom and J. Widengren, Physics 3, 40 (2010).
    [CrossRef]
  3. C. J. R. Sheppard, Optik 80, 53 (1988).
  4. A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
    [CrossRef]
  5. G. De Luca, R. Breedijk, and E. Manders, “Point-scanning based super-resolution microscopy,” presented at Focus on Microscopy, Maastricht, 2013.
  6. W. Lukosz and M. Marchand, Optica Acta 10, 241 (1963).
    [CrossRef]
  7. M. M. A. Neil, R. Juskaitis, and T. Wilson, Opt. Lett. 22, 1905 (1997).
    [CrossRef]
  8. M. Gustafsson, J. Microsc. 198, 82 (2000).
    [CrossRef]
  9. R. Heintzmann and P. Benedetti, “Space and frequency domain reconstruction strategies in a confocal microscope based on patterned illumination and wide-field image collection,” presented at Focus on Microscopy, Genoa, 2003.
  10. I. J. Cox, C. J. R. Sheppard, and T. Wilson, Appl. Opt. 21, 778 (1982).
    [CrossRef]
  11. C. J. R. Sheppard and H. J. Matthews, J. Mod. Opt. 35, 145 (1988).
    [CrossRef]
  12. T. Wilson and A. R. Carlini, Appl. Opt. 27, 3791 (1988).
    [CrossRef]
  13. M. Born and E. Wolf, Principles of Optics, 5th ed. (Pergamon, 1975).
  14. S. B. Mehta and R. Oldenbourg, “microlith: image simulation for biological phase microscopy,” arXiv:1305.7149v1.
  15. I. J. Cox, C. J. R. Sheppard, and T. Wilson, Optik 60, 391 (1982).
  16. G. Cox and C. J. R. Sheppard, Microsc. Res. Tech. 63, 18 (2004).
    [CrossRef]
  17. R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
    [CrossRef]
  18. S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” arXiv: 1306.6230.

2012

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

2010

C. B. Müller and J. Enderlein, Phys. Rev. Lett. 104, 198101 (2010).
[CrossRef]

H. Blom and J. Widengren, Physics 3, 40 (2010).
[CrossRef]

2004

G. Cox and C. J. R. Sheppard, Microsc. Res. Tech. 63, 18 (2004).
[CrossRef]

2003

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

2000

M. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef]

1997

1988

C. J. R. Sheppard and H. J. Matthews, J. Mod. Opt. 35, 145 (1988).
[CrossRef]

T. Wilson and A. R. Carlini, Appl. Opt. 27, 3791 (1988).
[CrossRef]

C. J. R. Sheppard, Optik 80, 53 (1988).

1982

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Optik 60, 391 (1982).

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Appl. Opt. 21, 778 (1982).
[CrossRef]

1963

W. Lukosz and M. Marchand, Optica Acta 10, 241 (1963).
[CrossRef]

Benedetti, P.

R. Heintzmann and P. Benedetti, “Space and frequency domain reconstruction strategies in a confocal microscope based on patterned illumination and wide-field image collection,” presented at Focus on Microscopy, Genoa, 2003.

Blom, H.

H. Blom and J. Widengren, Physics 3, 40 (2010).
[CrossRef]

Born, M.

M. Born and E. Wolf, Principles of Optics, 5th ed. (Pergamon, 1975).

Breedijk, R.

G. De Luca, R. Breedijk, and E. Manders, “Point-scanning based super-resolution microscopy,” presented at Focus on Microscopy, Maastricht, 2013.

Carlini, A. R.

Chitnis, A. B.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Combes, C. A.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Cox, G.

G. Cox and C. J. R. Sheppard, Microsc. Res. Tech. 63, 18 (2004).
[CrossRef]

Cox, I. J.

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Optik 60, 391 (1982).

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Appl. Opt. 21, 778 (1982).
[CrossRef]

Enderlein, J.

C. B. Müller and J. Enderlein, Phys. Rev. Lett. 104, 198101 (2010).
[CrossRef]

Fischer, R. S.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Gustafsson, M.

M. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef]

Hanley, Q. S.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Heintzmann, R.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” arXiv: 1306.6230.

R. Heintzmann and P. Benedetti, “Space and frequency domain reconstruction strategies in a confocal microscope based on patterned illumination and wide-field image collection,” presented at Focus on Microscopy, Genoa, 2003.

Jovin, T. M.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Juskaitis, R.

Luca, G. De

G. De Luca, R. Breedijk, and E. Manders, “Point-scanning based super-resolution microscopy,” presented at Focus on Microscopy, Maastricht, 2013.

Lukosz, W.

W. Lukosz and M. Marchand, Optica Acta 10, 241 (1963).
[CrossRef]

Manders, E.

G. De Luca, R. Breedijk, and E. Manders, “Point-scanning based super-resolution microscopy,” presented at Focus on Microscopy, Maastricht, 2013.

Marchand, M.

W. Lukosz and M. Marchand, Optica Acta 10, 241 (1963).
[CrossRef]

Matthews, H. J.

C. J. R. Sheppard and H. J. Matthews, J. Mod. Opt. 35, 145 (1988).
[CrossRef]

Mehta, S. B.

S. B. Mehta and R. Oldenbourg, “microlith: image simulation for biological phase microscopy,” arXiv:1305.7149v1.

Mione, M.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Müller, C. B.

C. B. Müller and J. Enderlein, Phys. Rev. Lett. 104, 198101 (2010).
[CrossRef]

Munroe, P.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Nailon, J.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Neil, M. 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. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Oldenbourg, R.

S. B. Mehta and R. Oldenbourg, “microlith: image simulation for biological phase microscopy,” arXiv:1305.7149v1.

Parekh, S. H.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Roth, S.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” arXiv: 1306.6230.

Sarafis, V.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Sheppard, C. J. R.

G. Cox and C. J. R. Sheppard, Microsc. Res. Tech. 63, 18 (2004).
[CrossRef]

C. J. R. Sheppard, Optik 80, 53 (1988).

C. J. R. Sheppard and H. J. Matthews, J. Mod. Opt. 35, 145 (1988).
[CrossRef]

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Appl. Opt. 21, 778 (1982).
[CrossRef]

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Optik 60, 391 (1982).

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” arXiv: 1306.6230.

Shroff, H.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Temprine, K.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Wicker, K.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” arXiv: 1306.6230.

Widengren, J.

H. Blom and J. Widengren, Physics 3, 40 (2010).
[CrossRef]

Wilson, T.

Wolf, E.

M. Born and E. Wolf, Principles of Optics, 5th ed. (Pergamon, 1975).

York, A. G.

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Appl. Opt.

J. Microsc.

M. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef]

J. Mod. Opt.

C. J. R. Sheppard and H. J. Matthews, J. Mod. Opt. 35, 145 (1988).
[CrossRef]

Micron

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, Micron 34, 293 (2003).
[CrossRef]

Microsc. Res. Tech.

G. Cox and C. J. R. Sheppard, Microsc. Res. Tech. 63, 18 (2004).
[CrossRef]

Nat. Methods

A. G. York, S. H. Parekh, D. D. Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combes, and H. Shroff, Nat. Methods 9, 749 (2012).
[CrossRef]

Opt. Lett.

Optica Acta

W. Lukosz and M. Marchand, Optica Acta 10, 241 (1963).
[CrossRef]

Optik

C. J. R. Sheppard, Optik 80, 53 (1988).

I. J. Cox, C. J. R. Sheppard, and T. Wilson, Optik 60, 391 (1982).

Phys. Rev. Lett.

C. B. Müller and J. Enderlein, Phys. Rev. Lett. 104, 198101 (2010).
[CrossRef]

Physics

H. Blom and J. Widengren, Physics 3, 40 (2010).
[CrossRef]

Other

G. De Luca, R. Breedijk, and E. Manders, “Point-scanning based super-resolution microscopy,” presented at Focus on Microscopy, Maastricht, 2013.

R. Heintzmann and P. Benedetti, “Space and frequency domain reconstruction strategies in a confocal microscope based on patterned illumination and wide-field image collection,” presented at Focus on Microscopy, Genoa, 2003.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” arXiv: 1306.6230.

M. Born and E. Wolf, Principles of Optics, 5th ed. (Pergamon, 1975).

S. B. Mehta and R. Oldenbourg, “microlith: image simulation for biological phase microscopy,” arXiv:1305.7149v1.

Supplementary Material (1)

» Media 1: AVI (3246 KB)     

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Figures (5)

Fig. 1.
Fig. 1.

(a) Cross-section through the image of a point object in a scanning microscope for a point detector displaced a normalized distance vd from the axis. There is assumed to be no Stokes shift. The peak intensity is reduced and shifted sideways as the offset is increased, (b) after shifting the image so that its peak is on the axis, and (c) cross-section through the (circularly symmetric) image of a point object in a scanning microscope after pixel reassignment with a detector array of normalized radius vdmax. The conventional case is also shown. (d) Peak intensity and normalized half-width of the image of a point object after pixel reassignment with a detector array of normalized radius vdmax. The half-width is normalized to unity for a conventional microscope. The detection efficiency (normalized to unity for the conventional case) is also shown.

Fig. 2.
Fig. 2.

3D OTFs for (a) conventional fluorescence and (b) after pixel reassignment. There is assumed to be no Stokes shift.

Fig. 3.
Fig. 3.

Effective PSF for different pixels on a 9×9 detector array of size 9λ/NA, just larger than the first zero of the Airy disc. (a) Stokes shift is neglected. (b) Stokes shift is assumed to be such that λem/λex=1.5. The central pixel gives close to a true confocal image.

Fig. 4.
Fig. 4.

Frame from Media 1: transverse PSF due to summation of effective PSFs shown in Fig. 3(b) without and with reassignment. Spatial coordinates are expressed in terms of v=(2π/λex)nxsinα.

Fig. 5.
Fig. 5.

(a) Comparison of the transverse and axial PSFs for a conventional microscope, and for summation over a 9×9 pixel detector array, without and with reassignment at a weighting factor of one. (b) Transverse projection of the 3D OTFs obtained by 2D Fourier transforms of PSFs shown in (a). The display gamma is 0.5. Note that the transverse and axial axes are switched between PSF and OTF plots. Distance and spatial-frequencies are expressed in normalized coordinates of v=(2π/λex)nxsinα and m=nxsinα/λex, respectively in terms of the excitation wavelength.

Tables (1)

Tables Icon

Table 1. Imaging Performance with Pixel Reassignment Compared with Conventional and Confocal Microscopes

Equations (5)

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

I(xs,xd)=H1(x)F(xxs)H2(xxd)d2x,
I(x1,x2)=H1(xx1)H2(xx2)F(x)d2x.
I(xr)=H1[xxraxd]H2[xxr+(1a)xd]×F(x)S(xd)d2xd2xd.
Ceff(m)=C1[(1a)m]C2[am]
Ceff(m)=C1(βm1+β)C2(m1+β).

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