Abstract

Structured illumination has been employed in fluorescence microscopy to extend its lateral resolution. It has been demonstrated that a factor of 2 improvement can be achieved. In this paper, we introduce a novel optical arrangement that can further improve the resolution. It makes use of a fine grating held in close proximity to the sample. The fringe pattern thus projected onto the sample contains grating vectors substantially higher than those that are possible with the conventional structured illumination setup. We will present experimental results to demonstrate the principle of the technique, and will show that, theoretically, it can achieve an imaging NA approaching 4.

© 2010 Optical Society of America

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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, 82–87 (2000).
    [CrossRef] [PubMed]
  3. S. W. Hell and Jan Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19, 780–782(1994).
    [CrossRef] [PubMed]
  4. S. W. Hell, E. H. K. Stelzer, S. Lindek, and C. Cremer, “Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy,” Opt. Lett. 19, 222–224 (1994).
    [CrossRef] [PubMed]
  5. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100nm axial resolution,” J. Microsc. 195, 10–16 (1999).
    [CrossRef] [PubMed]
  6. B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
    [CrossRef] [PubMed]
  7. M. G. Somekh, K. Hsu, and M. C. Pitter, “Resolution in structured illumination microscopy: a probabilistic approach,” J. Opt. Soc. Am. 25, 1319–1329 (2008).
    [CrossRef]
  8. S. Liu, C. J. Chuang, C. W. See, G. Zoriniants, W. L. Barnes, and M. G. Somekh, “Double-grating-structured light microscope using plasmonic nanoparticle arrays,” Opt. Lett. 34, 1255–1257 (2009).
    [CrossRef] [PubMed]
  9. The value of the k-vector is normalized with respect to 2π/λ so that it has a maximum value of 1 in air.
  10. A. Sentenac, K. Belkebir, H. Giovannini, and P. C. Chaumet, “Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate,” Opt. Lett. 33, 255–257 (2008).
    [CrossRef] [PubMed]
  11. J. W. Goodman, Introduction to Fourier Optics (Roberts & Co., 2004).
  12. F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
    [CrossRef]
  13. J. K. Consulting, “Thin film design and applications,” http://www.kruschwitz.com/materials.htm.

2009 (1)

2008 (2)

A. Sentenac, K. Belkebir, H. Giovannini, and P. C. Chaumet, “Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate,” Opt. Lett. 33, 255–257 (2008).
[CrossRef] [PubMed]

M. G. Somekh, K. Hsu, and M. C. Pitter, “Resolution in structured illumination microscopy: a probabilistic approach,” J. Opt. Soc. Am. 25, 1319–1329 (2008).
[CrossRef]

2006 (1)

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

2000 (1)

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]

1999 (2)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[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]

1994 (2)

1993 (1)

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
[CrossRef] [PubMed]

Agard, D. A.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[CrossRef] [PubMed]

Bailey, B.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
[CrossRef] [PubMed]

Barnes, W. L.

Belkebir, K.

Chaumet, P. C.

Chuang, C. J.

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]

S. W. Hell, E. H. K. Stelzer, S. Lindek, and C. Cremer, “Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy,” Opt. Lett. 19, 222–224 (1994).
[CrossRef] [PubMed]

Espinos, J. P.

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Farkas, D. L.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
[CrossRef] [PubMed]

Giovannini, H.

Girardeau, T.

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Gonzalez-Elipe, A. R.

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Goodman, J. W.

J. W. Goodman, Introduction to Fourier Optics (Roberts & Co., 2004).

Gracia, F.

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Gustafsson, M. G. L.

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]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[CrossRef] [PubMed]

Heintzmann, R.

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

Hell, S. W.

Holgado, J. P.

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Hsu, K.

M. G. Somekh, K. Hsu, and M. C. Pitter, “Resolution in structured illumination microscopy: a probabilistic approach,” J. Opt. Soc. Am. 25, 1319–1329 (2008).
[CrossRef]

Lanni, F.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
[CrossRef] [PubMed]

Lindek, S.

Liu, S.

Pitter, M. C.

M. G. Somekh, K. Hsu, and M. C. Pitter, “Resolution in structured illumination microscopy: a probabilistic approach,” J. Opt. Soc. Am. 25, 1319–1329 (2008).
[CrossRef]

Sedat, J. W.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[CrossRef] [PubMed]

See, C. W.

Sentenac, A.

Somekh, M. G.

S. Liu, C. J. Chuang, C. W. See, G. Zoriniants, W. L. Barnes, and M. G. Somekh, “Double-grating-structured light microscope using plasmonic nanoparticle arrays,” Opt. Lett. 34, 1255–1257 (2009).
[CrossRef] [PubMed]

M. G. Somekh, K. Hsu, and M. C. Pitter, “Resolution in structured illumination microscopy: a probabilistic approach,” J. Opt. Soc. Am. 25, 1319–1329 (2008).
[CrossRef]

Stelzer, E. H. K.

Taylor, D. L.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
[CrossRef] [PubMed]

Wichmann, Jan

Yubero, F.

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Zoriniants, G.

J. Microsc. (2)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[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).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

M. G. Somekh, K. Hsu, and M. C. Pitter, “Resolution in structured illumination microscopy: a probabilistic approach,” J. Opt. Soc. Am. 25, 1319–1329 (2008).
[CrossRef]

Nature (1)

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, 44–48 (1993).
[CrossRef] [PubMed]

Opt. Lett. (4)

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]

Thin Solid Films (1)

F. Gracia, F. Yubero, J. P. Holgado, J. P. Espinos, A. R. Gonzalez-Elipe, and T. Girardeau, “SiO2/TiO2 thin films with variable refractive index prepared by ion beaminduced and plasma enhanced chemical vapour deposition,” Thin Solid Films 500, 19–26 (2006).
[CrossRef]

Other (3)

J. K. Consulting, “Thin film design and applications,” http://www.kruschwitz.com/materials.htm.

The value of the k-vector is normalized with respect to 2π/λ so that it has a maximum value of 1 in air.

J. W. Goodman, Introduction to Fourier Optics (Roberts & Co., 2004).

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Figures (8)

Fig. 1
Fig. 1

Sample unit showing the principle of the technique.

Fig. 2
Fig. 2

Combining the recovered virtual apertures to form an extended image spectrum.

Fig. 3
Fig. 3

System configuration including the grating pattern used in the sample unit.

Fig. 4
Fig. 4

a, Intensity pattern of the grating projected onto the sample plane, and b, Fourier components of the intensity pattern in (a).

Fig. 5
Fig. 5

Sample unit showing the fluorescent beads. The thin film used in the experiment is 75 μm thick microscope immersion oil.

Fig. 6
Fig. 6

a, Image of fluorescent beads using reduced aperture ( NA = 0.17 ); b, reconstructed image using the zero, ± 1 , and ± 2 orders; and c, image obtained using increased system aperture ( NA 0.8 ).

Fig. 7
Fig. 7

Intensity profiles of the fluorescent bead labeled p in Fig. 6b. Profile 1: obtained using the reduced NA of 0.17; profile 2: reconstructed using the zero and ± 1 orders; profile 3: reconstructed using the zero, ± 1 , and ± 2 orders. Dashed curves are simulated profiles using NA of 0.17, 0.28, and 0.37, respectively, for the three profiles.

Fig. 8
Fig. 8

Computer simulations of psfs using square (row a) and triangular (row b) grating patterns. Column 2 shows locations of the virtual apertures; column 3 provides the intensity psfs. Also shown in column 3 is the psf (dashed curve) obtained using the ideal large aperture (large dashed circle in column 2).

Tables (1)

Tables Icon

Table 1 Simulated Intensity psf using Square and Triangular Grating Patterns a

Equations (8)

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I ( x ) = 1 + g 1 cos ( k 1 x + θ 1 ) + g 2 cos ( 2 k 1 x + θ 2 ) + ,
O m ( x ) = [ I m ( x ) S ( x ) ] H ( x ) ,
O ^ m ( f x ) = S ^ m ( f x ) H ^ ( f x ) + 1 2 S ^ m ( f x f g ) H ^ ( f x ) exp j θ + 1 2 S ^ m ( f x + f g ) H ^ ( f g ) exp j θ + 1 2 S ^ m ( f x 2 f g ) H ^ ( f x ) exp j 2 θ + 1 2 S ^ m ( f x + 2 f g ) H ^ ( f x ) exp j 2 θ + ,
N = 2 M + 1.
k g = n 2 π λ o ,
T g = λ o n .
f p g = 2 f g + 2 f 0 = 2 n λ 0 + 2 NA λ 0 = 2 λ 0 ( n + NA ) = 2 λ 0 NA eff ,
T 0 = m λ 0 n ,

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