Abstract

We developed a structured illumination microscopy (SIM) system that uses a spatial light modulator (SLM) to generate interference illumination patterns at four orientations - 0°, 45°, 90°, and 135°, to reconstruct a high-resolution image. The use of a SLM for pattern alterations is rapid and precise, without mechanical calibration; moreover, our design of SLM patterns allows generating the four illumination patterns of high contrast and nearly equivalent periods to achieve a near isotropic enhancement in lateral resolution. We compare the conventional image of 100-nm beads with those reconstructed from two (0°+90° or 45°+135°) and four (0°+45°+90°+135°) pattern orientations to show the differences in resolution and image, with the support of simulations. The reconstructed images of 200-nm beads at various depths and fine structures of actin filaments near the edge of a HeLa cell are presented to demonstrate the intensity distributions in the axial direction and the prospective application to biological systems.

© 2009 Optical Society of America

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  1. R. Heintzmann, and G. Ficz, "Breaking the resolution limit in light microscopy," Brief. Funct. Genomics Proteomics 5, 289-301 (2006).
  2. M. G. L. Gustafsson, "Extended resolution fluorescence microscopy," Curr. Opin. Struct. Biol. 9, 627-634 (1999).
    [PubMed]
  3. B. Balley, 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).
  4. R. Heintzmann, and C. Cremer, "Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating," Proc. SPIE 3568, 185-196 (1998).
  5. M. G. L. Gustafsson, "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy," J. Microsc. 198, 82-87 (2000).
    [PubMed]
  6. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination," Proc. SPIE 3919, 141-150 (2000).
  7. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "I5M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microsc. 195, 10-16 (1999).
    [PubMed]
  8. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).
  9. 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. U. S. A. 97, 7232-7236 (2000).
    [PubMed]
  10. S. W. Hell, S. Lindek, C. Cremer, and E. H. K. Stelzer, "Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution," Appl. Phys. Lett. 64, 1335-1337 (1994).
  11. T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
    [PubMed]
  12. 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).
    [PubMed]
  13. M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
    [PubMed]
  14. R. Heintzmann, and T. M. Jovin, "Saturated patterned excitation microscopy-a concept for optical resolution improvement," J. Opt. Soc. Am. A 19, 1599-1609 (2002).
  15. R. Heintzmann, "Saturated patterned excitation microscopy with two-dimensional excitation patterns," Micron 34, 283-291 (2003).
    [PubMed]
  16. M. R. Beversluis, G. W. Bryant, and S. J. Stranick, "Effects of inhomogeneous fields in superresolving structured-illumination microscopy," J. Opt. Soc. Am. A 25, 1371-1377 (2008).
  17. E. Chung, D. Kim, and P. T. C. So, "Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy," Opt. Lett. 31, 945-947 (2006).
    [PubMed]
  18. R. Fiolka, M. Beck, and A. Stemmer, "Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator," Opt. Lett. 33, 1629-1631 (2008).
    [PubMed]
  19. M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
    [PubMed]
  20. L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
    [PubMed]
  21. J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

2008 (4)

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
[PubMed]

M. R. Beversluis, G. W. Bryant, and S. J. Stranick, "Effects of inhomogeneous fields in superresolving structured-illumination microscopy," J. Opt. Soc. Am. A 25, 1371-1377 (2008).

R. Fiolka, M. Beck, and A. Stemmer, "Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator," Opt. Lett. 33, 1629-1631 (2008).
[PubMed]

2006 (4)

E. Chung, D. Kim, and P. T. C. So, "Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy," Opt. Lett. 31, 945-947 (2006).
[PubMed]

J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

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).
[PubMed]

R. Heintzmann, and G. Ficz, "Breaking the resolution limit in light microscopy," Brief. Funct. Genomics Proteomics 5, 289-301 (2006).

2005 (1)

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
[PubMed]

2003 (1)

R. Heintzmann, "Saturated patterned excitation microscopy with two-dimensional excitation patterns," Micron 34, 283-291 (2003).
[PubMed]

2002 (1)

2000 (4)

T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
[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. U. S. A. 97, 7232-7236 (2000).
[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).
[PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination," Proc. SPIE 3919, 141-150 (2000).

1999 (2)

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

M. G. L. Gustafsson, "Extended resolution fluorescence microscopy," Curr. Opin. Struct. Biol. 9, 627-634 (1999).
[PubMed]

1998 (1)

R. Heintzmann, and C. Cremer, "Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating," Proc. SPIE 3568, 185-196 (1998).

1995 (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).

1994 (1)

S. W. Hell, S. Lindek, C. Cremer, and E. H. K. Stelzer, "Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution," Appl. Phys. Lett. 64, 1335-1337 (1994).

1993 (1)

B. Balley, 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).

Agard, D. A.

L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
[PubMed]

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination," Proc. SPIE 3919, 141-150 (2000).

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

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).

Balley, B.

B. Balley, 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).

Beck, M.

Betzig, E.

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).
[PubMed]

Beversluis, M. R.

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).
[PubMed]

Bryant, G. W.

Cande, W. Z.

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

Cariton, P. M.

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

Chung, 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 (1998).

S. W. Hell, S. Lindek, C. Cremer, and E. H. K. Stelzer, "Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution," Appl. Phys. Lett. 64, 1335-1337 (1994).

Davidson, M. W.

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).
[PubMed]

Dyba, M.

T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
[PubMed]

Egner, A.

T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
[PubMed]

Farkas, D. L.

B. Balley, 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).

Ficz, G.

R. Heintzmann, and G. Ficz, "Breaking the resolution limit in light microscopy," Brief. Funct. Genomics Proteomics 5, 289-301 (2006).

Fiolka, R.

Freeman, D. M.

J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

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. U. S. A. 97, 7232-7236 (2000).
[PubMed]

Golubovskaya, I. N.

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
[PubMed]

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
[PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination," Proc. SPIE 3919, 141-150 (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).
[PubMed]

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

M. G. L. Gustafsson, "Extended resolution fluorescence microscopy," Curr. Opin. Struct. Biol. 9, 627-634 (1999).
[PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).

Heintzmann, R.

R. Heintzmann, and G. Ficz, "Breaking the resolution limit in light microscopy," Brief. Funct. Genomics Proteomics 5, 289-301 (2006).

R. Heintzmann, "Saturated patterned excitation microscopy with two-dimensional excitation patterns," Micron 34, 283-291 (2003).
[PubMed]

R. Heintzmann, and T. M. Jovin, "Saturated patterned excitation microscopy-a concept for optical resolution improvement," J. Opt. Soc. Am. A 19, 1599-1609 (2002).

R. Heintzmann, and C. Cremer, "Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating," Proc. SPIE 3568, 185-196 (1998).

Hell, S. W.

T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
[PubMed]

S. W. Hell, S. Lindek, C. Cremer, and E. H. K. Stelzer, "Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution," Appl. Phys. Lett. 64, 1335-1337 (1994).

Hess, H. F.

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).
[PubMed]

Hong, S. S.

J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

Horn, B. K. P.

J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

Isaac, B.

L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
[PubMed]

Jacobs, S.

T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
[PubMed]

Jovin, T. M.

Kim, D.

Klar, T. A.

T. A. Klar, S. Jacobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sci. U. S. A. 97, 8206-8210 (2000).
[PubMed]

Knapp, H. F.

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. U. S. A. 97, 7232-7236 (2000).
[PubMed]

Lanni, F.

B. Balley, 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).

Lindek, S.

S. W. Hell, S. Lindek, C. Cremer, and E. H. K. Stelzer, "Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution," Appl. Phys. Lett. 64, 1335-1337 (1994).

Lindwasser, O. W.

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).
[PubMed]

Lippincott-Schwartz, J.

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).
[PubMed]

Mermelstein, M. S.

J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

Olenych, 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).
[PubMed]

Patterson, G. H.

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).
[PubMed]

Ryu, J.

J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171112-3 (2006).

Sedat, J. W.

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
[PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination," Proc. SPIE 3919, 141-150 (2000).

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

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).

Shao, L.

M. G. L. Gustafsson, L. Shao, P. M. Cariton, 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, 4957-4970 (2008).
[PubMed]

L. Shao, B. Isaac, S. Uzawa, D. A. Agard, J. W. Sedat, and M. G. L. Gustafsson, "I5S: Wide-field light microscopy with 100-nm-Scale resolution in three dimensions," Biophys. J. 94, 4971-4983 (2008).
[PubMed]

So, P. T. C.

Sougrat, R.

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).
[PubMed]

Stelzer, E. H. K.

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Figures (8)

Fig. 1.
Fig. 1.

Shifts of D′(k-k0) and D′(k+k0) to D(k-k0) and D(k+k0) and their addition to D(k) to form the final spectrum acquired with 0° illumination pattern at three phases

Fig. 2.
Fig. 2.

SI microscope setup

Fig. 3.
Fig. 3.

0° and 45° SLM patterns at three phases and their corresponding illumination patterns; the black ones in the SLM patterns indicate no phase retardation on the incident beam

Fig. 4.
Fig. 4.

(a-d) Simulations of images and intensity distributions of 100-nm beads for conventional and structured illumination microscopy reconstructed from 0°+90°, 45°+135° and 0°+90°+45°+135° pattern orientations; the solid lines represent Gaussian-fits and the brightness and contrasts were adjusted to show side lobes clearly

Fig. 5.
Fig. 5.

(a-d) Experimental spectra of spatial frequency, images and intensity distributions of 100-nm beads for conventional and structured illumination microscopy reconstructed from 0°+90°, 45°+135° and 0°+90°+45°+135° pattern orientations, without additions of a Wiener filter and an apodization function; the solid lines represent Gaussian-fits

Fig. 6.
Fig. 6.

(a, b) In-focus images of 200-nm beads for conventional and structured-illumination microscopy, respectively, (c, d) corresponding axial images of the blue region in Figs. 6(a) and 6(b), taken at a step 100 nm for each section, and (e) intensity profiles along the red dotted lines

Fig. 7.
Fig. 7.

SI image of a dye-labeled actin cytoskeleton at the edge of a HeLa cell with (a) 0°+90°, (b) 45°+135°, (c) 0°+90°+45°+135° reconstructions, and (d) a Wiener filter and an apodization function on image (c) to show the differences in resolution resulting from the reconstructions of separate pattern orientations

Fig. 8.
Fig. 8.

(a) Conventional image of a dye-labeled actin cytoskeleton, (b) the corresponding SI image after Wiener filter and apodization treatment for comparison, and (c) the intensity distributions along the dotted lines to show the well resolved structures separated by 186 nm in SI image

Equations (6)

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

D(r)=(S(r)I(r))P(r),
D(k)=(S(k)I(k))OTF(k),
I(r)=I0[1+cos(k0·r+ϕ)],
D(k)=I0(S(k)+0.5S(k+k0)eiϕ+0.5S(kk0)eiϕ)OTF(k),
[D1(k)D2(k)D3(k)]=I0[10.5eiϕ10.5eiϕ110.5eiϕ20.5eiϕ210.5eiϕ30.5eiϕ3][D(k)D(k+k0)D(k+k0)].
S(k)=SD(k)SOTF(k)+w2,

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