Abstract

We discuss data treatment strategies in structured illumination microscopy, using simulated and experimental data. In the setup, the illumination pattern is generated by projecting a movable pinhole mask into the sample, and a wide-field fluorescence microscope image is acquired for each pattern position. The structured illumination data obtained from a two-dimensional illumination pattern can be treated by projection strategies such as in video confocal microscopy (sum, maximum, maximum minus minimum, and superconfocal), by a scaled subtraction of the out-of-focus estimate, or by a modified version of the Fourier-space treatment, as is known for data from one-dimensional structured illumination. We investigate the influence of some data processing strategies on unwanted effects such as residual patterning and local deviations from linearity in the reconstructed intensity.

© 2006 Optical Society of America

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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
  22. W. Lukosz and M. Marchand, "Optischen Abbildung unter Überschreitung der beugungsbedingten Auflösungsgrenze," Opt. Acta 10, 241-255 (1963).
    [CrossRef]
  23. W. Lukosz, "Optical systems with resolving powers exceeding the classical limit. II," J. Opt. Soc. Am. 57, 932-941 (1967).
    [CrossRef]
  24. A. Shemer, D. Mendlovic, Z. Zalevsky, J. Garcia, and P. Garcia Martinez, "Superresolving optical system with time multiplexing and computer decoding," Appl. Opt. 38, 7245-7251 (1999).
    [CrossRef]
  25. A. Egner and S. W. Hell, "Equivalence of the Huygens-Fresnel and Debeye approach for the calculation of high aperture point-spread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999).
    [CrossRef]
  26. M. G. L. Gustafsson, Department of Physiology and Division of Bioengineering, University of California, San Francisco, Room GH-N412B, Box 2240, 600 16th Street, San Francisco, Calif. 94143-2240 (personal communication, 2001).

2003

2002

2001

2000

A. L. P. Dlugan, C. E. MacAulay, and P. M. Lane, "Improvements to quantitative microscopy through the use of digital micromirror devices," in Optical Diagnostics of Living Cells III, D. L. Farkas and R. C. Leif, eds., Proc. SPIE 3921, 6-11 (2000).
[CrossRef]

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. USA 97, 7232-7236 (2000).
[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]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination microscopy," in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VII, J.-A. Conchello, C. J. Cogswell, A. G. Tescher, and T. Wilson, eds., Proc. SPIE 3919, 141-150 (2000).
[CrossRef]

1999

R. Heintzmann and C. Cremer, "Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating," in Optical Biopsies and Microscopic Techniques III, I. J. Bigio, H. Schneckenburger, J. Slavik, K. Svanberg, and P. M. Viallet, eds., Proc. SPIE 3568, 185-196 (1999).
[CrossRef]

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

A. Shemer, D. Mendlovic, Z. Zalevsky, J. Garcia, and P. Garcia Martinez, "Superresolving optical system with time multiplexing and computer decoding," Appl. Opt. 38, 7245-7251 (1999).
[CrossRef]

A. Egner and S. W. Hell, "Equivalence of the Huygens-Fresnel and Debeye approach for the calculation of high aperture point-spread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999).
[CrossRef]

1998

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
[CrossRef]

1997

1995

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson and C. J. Cogswell, eds., Proc. SPIE 2412, 56-62 (1995).
[CrossRef]

1994

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Achieving confocal point performance in confocal line microscopy," Bioimaging 2, 122-130 (1994).
[CrossRef]

1968

1967

1963

W. Lukosz and M. Marchand, "Optischen Abbildung unter Überschreitung der beugungsbedingten Auflösungsgrenze," Opt. Acta 10, 241-255 (1963).
[CrossRef]

Agard, D. A.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination microscopy," in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VII, J.-A. Conchello, C. J. Cogswell, A. G. Tescher, and T. Wilson, eds., Proc. SPIE 3919, 141-150 (2000).
[CrossRef]

M. G. L. Gustafsson, J. W. Sedat, and D. A. Agard, "Method and apparatus for three-dimensional microscopy with enhanced depth resolution," U.S. patent 5,671,085 (23 September 1997).

Andresen, V.

Arndt-Jovin, D.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, "A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images," J. Microsc. 204, 119-137 (2001).
[CrossRef] [PubMed]

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

Benedetti, P. A.

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson and C. J. Cogswell, eds., Proc. SPIE 2412, 56-62 (1995).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Achieving confocal point performance in confocal line microscopy," Bioimaging 2, 122-130 (1994).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Method for the acquisition of images by confocal microscopy," U.S. patent 6,016,367 (18 January 2000).

Bewersdorf, J.

Cremer, C.

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

R. Heintzmann and C. Cremer, "Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating," in Optical Biopsies and Microscopic Techniques III, I. J. Bigio, H. Schneckenburger, J. Slavik, K. Svanberg, and P. M. Viallet, eds., Proc. SPIE 3568, 185-196 (1999).
[CrossRef]

R. Heintzmann and C. Cremer, "Method and device for representing an object," U.S. patent 6,909,105 (21 June 2005).

Dlugan, A. L. P.

A. L. P. Dlugan, C. E. MacAulay, and P. M. Lane, "Improvements to quantitative microscopy through the use of digital micromirror devices," in Optical Diagnostics of Living Cells III, D. L. Farkas and R. C. Leif, eds., Proc. SPIE 3921, 6-11 (2000).
[CrossRef]

Egger, M. D.

Egner, A.

V. Andresen, A. Egner, and S. W. Hell, "Time-multiplexed multifocal multiphoton microscope," Opt. Lett. 26, 75-77 (2001).
[CrossRef]

A. Egner and S. W. Hell, "Equivalence of the Huygens-Fresnel and Debeye approach for the calculation of high aperture point-spread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999).
[CrossRef]

Evangelista, V.

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson and C. J. Cogswell, eds., Proc. SPIE 2412, 56-62 (1995).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Achieving confocal point performance in confocal line microscopy," Bioimaging 2, 122-130 (1994).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Method for the acquisition of images by confocal microscopy," U.S. patent 6,016,367 (18 January 2000).

Frohn, J. T.

J. T. Frohn, H. F. Knapp, and A. Stemmer, "Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation," Opt. Lett. 26, 828-830 (2001).
[CrossRef]

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. USA 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Fukano, T.

Galambos, R.

Garcia, J.

Gemkov, M. J.

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

Guidarini, D.

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson and C. J. Cogswell, eds., Proc. SPIE 2412, 56-62 (1995).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Achieving confocal point performance in confocal line microscopy," Bioimaging 2, 122-130 (1994).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Method for the acquisition of images by confocal microscopy," U.S. patent 6,016,367 (18 January 2000).

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, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination microscopy," in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VII, J.-A. Conchello, C. J. Cogswell, A. G. Tescher, and T. Wilson, eds., Proc. SPIE 3919, 141-150 (2000).
[CrossRef]

M. G. L. Gustafsson, J. W. Sedat, and D. A. Agard, "Method and apparatus for three-dimensional microscopy with enhanced depth resolution," U.S. patent 5,671,085 (23 September 1997).

M. G. L. Gustafsson, Department of Physiology and Division of Bioengineering, University of California, San Francisco, Room GH-N412B, Box 2240, 600 16th Street, San Francisco, Calif. 94143-2240 (personal communication, 2001).

Hadravsky, M.

Hanley, Q. S.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, "A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images," J. Microsc. 204, 119-137 (2001).
[CrossRef] [PubMed]

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Heintzmann, R.

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

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

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, "A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images," J. Microsc. 204, 119-137 (2001).
[CrossRef] [PubMed]

R. Heintzmann and C. Cremer, "Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating," in Optical Biopsies and Microscopic Techniques III, I. J. Bigio, H. Schneckenburger, J. Slavik, K. Svanberg, and P. M. Viallet, eds., Proc. SPIE 3568, 185-196 (1999).
[CrossRef]

R. Heintzmann and C. Cremer, "Method and device for representing an object," U.S. patent 6,909,105 (21 June 2005).

Hell, S. W.

V. Andresen, A. Egner, and S. W. Hell, "Time-multiplexed multifocal multiphoton microscope," Opt. Lett. 26, 75-77 (2001).
[CrossRef]

A. Egner and S. W. Hell, "Equivalence of the Huygens-Fresnel and Debeye approach for the calculation of high aperture point-spread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999).
[CrossRef]

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
[CrossRef]

Jovin, T. M.

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

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, "A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images," J. Microsc. 204, 119-137 (2001).
[CrossRef] [PubMed]

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Juskaitis, R.

Knapp, H. F.

J. T. Frohn, H. F. Knapp, and A. Stemmer, "Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation," Opt. Lett. 26, 828-830 (2001).
[CrossRef]

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. USA 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Lane, P. M.

A. L. P. Dlugan, C. E. MacAulay, and P. M. Lane, "Improvements to quantitative microscopy through the use of digital micromirror devices," in Optical Diagnostics of Living Cells III, D. L. Farkas and R. C. Leif, eds., Proc. SPIE 3921, 6-11 (2000).
[CrossRef]

Lukosz, W.

W. Lukosz, "Optical systems with resolving powers exceeding the classical limit. II," J. Opt. Soc. Am. 57, 932-941 (1967).
[CrossRef]

W. Lukosz and M. Marchand, "Optischen Abbildung unter Überschreitung der beugungsbedingten Auflösungsgrenze," Opt. Acta 10, 241-255 (1963).
[CrossRef]

MacAulay, C. E.

A. L. P. Dlugan, C. E. MacAulay, and P. M. Lane, "Improvements to quantitative microscopy through the use of digital micromirror devices," in Optical Diagnostics of Living Cells III, D. L. Farkas and R. C. Leif, eds., Proc. SPIE 3921, 6-11 (2000).
[CrossRef]

Marchand, M.

W. Lukosz and M. Marchand, "Optischen Abbildung unter Überschreitung der beugungsbedingten Auflösungsgrenze," Opt. Acta 10, 241-255 (1963).
[CrossRef]

Martinez, P. Garcia

Mendlovic, D.

Miyawaki, A.

Neil, M. A. A.

Petran, M.

Pick, R.

Sedat, J. W.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination microscopy," in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VII, J.-A. Conchello, C. J. Cogswell, A. G. Tescher, and T. Wilson, eds., Proc. SPIE 3919, 141-150 (2000).
[CrossRef]

M. G. L. Gustafsson, J. W. Sedat, and D. A. Agard, "Method and apparatus for three-dimensional microscopy with enhanced depth resolution," U.S. patent 5,671,085 (23 September 1997).

Shemer, A.

Stemmer, A.

J. T. Frohn, H. F. Knapp, and A. Stemmer, "Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation," Opt. Lett. 26, 828-830 (2001).
[CrossRef]

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. USA 97, 7232-7236 (2000).
[CrossRef] [PubMed]

van Vliet, L. J.

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Verbeek, P. W.

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Verveer, P. J.

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Vestri, S.

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson and C. J. Cogswell, eds., Proc. SPIE 2412, 56-62 (1995).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Achieving confocal point performance in confocal line microscopy," Bioimaging 2, 122-130 (1994).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Method for the acquisition of images by confocal microscopy," U.S. patent 6,016,367 (18 January 2000).

Wilson, T.

Zalevsky, Z.

Appl. Opt.

Bioimaging

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Achieving confocal point performance in confocal line microscopy," Bioimaging 2, 122-130 (1994).
[CrossRef]

J. Microsc.

P. J. Verveer, Q. S. Hanley, P. W. Verbeek, L. J. van Vliet, and T. M. Jovin, "Theory of confocal fluorescence imaging in the Programmable Array Microscope (PAM)," J. Microsc. 189, 192-198 (1998).
[CrossRef]

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, and T. M. Jovin, "An optical sectioning programmable array microscope implemented with a digital micromirror device," J. Microsc. 196, 317-331 (1999).
[CrossRef] [PubMed]

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, "A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images," J. Microsc. 204, 119-137 (2001).
[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]

A. Egner and S. W. Hell, "Equivalence of the Huygens-Fresnel and Debeye approach for the calculation of high aperture point-spread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999).
[CrossRef]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

Micron

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

Opt. Acta

W. Lukosz and M. Marchand, "Optischen Abbildung unter Überschreitung der beugungsbedingten Auflösungsgrenze," Opt. Acta 10, 241-255 (1963).
[CrossRef]

Opt. Lett.

Proc. Natl. Acad. Sci. USA

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. USA 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Proc. SPIE

A. L. P. Dlugan, C. E. MacAulay, and P. M. Lane, "Improvements to quantitative microscopy through the use of digital micromirror devices," in Optical Diagnostics of Living Cells III, D. L. Farkas and R. C. Leif, eds., Proc. SPIE 3921, 6-11 (2000).
[CrossRef]

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Electronic multiconfocal points microscopy," in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson and C. J. Cogswell, eds., Proc. SPIE 2412, 56-62 (1995).
[CrossRef]

R. Heintzmann and C. Cremer, "Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating," in Optical Biopsies and Microscopic Techniques III, I. J. Bigio, H. Schneckenburger, J. Slavik, K. Svanberg, and P. M. Viallet, eds., Proc. SPIE 3568, 185-196 (1999).
[CrossRef]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination microscopy," in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VII, J.-A. Conchello, C. J. Cogswell, A. G. Tescher, and T. Wilson, eds., Proc. SPIE 3919, 141-150 (2000).
[CrossRef]

Other

M. G. L. Gustafsson, J. W. Sedat, and D. A. Agard, "Method and apparatus for three-dimensional microscopy with enhanced depth resolution," U.S. patent 5,671,085 (23 September 1997).

P. A. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, "Method for the acquisition of images by confocal microscopy," U.S. patent 6,016,367 (18 January 2000).

R. Heintzmann and C. Cremer, "Method and device for representing an object," U.S. patent 6,909,105 (21 June 2005).

M. G. L. Gustafsson, Department of Physiology and Division of Bioengineering, University of California, San Francisco, Room GH-N412B, Box 2240, 600 16th Street, San Francisco, Calif. 94143-2240 (personal communication, 2001).

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Figures (7)

Fig. 1
Fig. 1

Decomposed structure of the Fourier-transformed emitted light intensity. Dotted lines indicate the borders of the frequency region that can be transferred by the detection system of the fluorescence microscope. The emitted light is proportional to the product of the excitation light with the object's fluorophore distribution; in Fourier space this leads to a convolution. The object structure is attached to each of the delta-shaped peaks of the Fourier-transformed excitation structure. The emitted signal corresponds to the sum of these three object components.

Fig. 2
Fig. 2

Processing steps in PEM image reconstruction of experimental data (128 × 128 pixels of 250 nm × 250 nm each. Microscope, Leitz Orthoplan; objective, NPL Fluotar 40×, 1.3 NA; Pulnix 765; frame grabber, Redshift Imageworks. ∼450 nm excitation, ∼550 nm emission, 6 × 6 = 36 patterned images, 200 nm pixel pitch in sample; spot-to-spot distance, 2.1 μm in the sample). (a) Raw image; (b) fast Fourier transform of (a); (c) extracted zero order; (d) inverse fast Fourier transform of the zero order; (e) extracted higher order (k = −1, l = 0); (f) part (e) shifted such that the object's zero frequency aligns with the zero of the coordinate system; (g) all orders extracted, aligned, and added with weights; (h) reconstructed image, i.e., the inverse fast Fourier transform of (g). For clarity, the computational suppression of low-frequency information (see text) is not shown here.

Fig. 3
Fig. 3

Mouse kidney section from Molecular Probes (FluoCells #3; 36 μm × 36 μm). (a) Sum of the individual images. (b) Extracted and aligned third order. Here the zero-frequency region was suppressed to prevent the appearance of artifacts. (c) Maximum intensity projection. Note the residual patterning. (d) PEM reconstruction. Microscope, Leica DM-RXA2; objective, Leica HCX PL FL 100×, 1.3 NA; integrating analog camera, Pulnix 765; frame grabber, Scion LG-3.

Fig. 4
Fig. 4

Simulated comparison of methods. The simulation used a pinhole spacing of the illumination pinhole corresponding to 15 pixels of X and Y, respectively, and pinholes assumed to consist of a single pixel only. The pinhole array was stepped in a total of 5 × 5 = 25 steps. One pixel corresponds to 37.4 nm at an illumination wavelength of 488 nm. For simplicity, the same excitation PSF (NA, 1.4; 488 nm) that was calculated from vectorial diffraction theory[25] was also used as a fluorescence-emission PSF. (a) Object, clipped for better visualization; (b) example image; (c) average projection; (d) maximum intensity projection, Max; (e) Max − Min; Max + Min − 2 Avg; (g) PEM data processing.

Fig. 5
Fig. 5

Comparison of reconstructions from experimental data: (a) Avg, (b) Max, (c) Max − Min, (d) Max + Min − 2 Avg, (e) scaled subtraction with β = 1. (f) PEM reconstruction. For acquisition parameters see Fig. 2.

Fig. 6
Fig. 6

Simplified description of improvement in resolution for neighboring objects and loss of integral for the Max method of data processing. Rows 1–5, (left) steps in the illumination of one spot, indicated by an arrow and a dotted line, and (right) two neighboring spots. The illumination patterns (of relative strengths 0.25, 0.5, 1.0, 0.5, 0.25) with single-spot illumination are shown by dashed lines. Black and gray bars indicate the detected image of a sample assuming a similar detection PSF. The maximum projection, which is based on the gray bars, is shown gray, as is the shape of the sum projection (in black).

Fig. 7
Fig. 7

Image coding of relative positions, along (a) X and (b) Y, and (c) for total of the pixel to the nearest pinhole when this pixel showed maximal intensity in the sequence of pattern images. Note the bright flare about the object, especially at its edges, consistent with the situation indicated by the gray bar positions in phase step 2 of Fig. 6. For acquisition parameters see Fig. 2.

Equations (18)

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Avg ( x ) = i = 1 N img i ( x ) N
Max ( x ) = MAX i = 1 N [ img i ( x ) ] ,
Min ( x ) = MIN i = 1 N [ img i ( x ) ]
Super ( x ) = Max ( x ) + Min ( x ) 2   Avg ( x ) ,
ScaSub ( x , y ) = i = 1 N img i ( x , y ) Mask i o n ( x , y ) 1 γ i = 1 N img i ( x , y ) M ask i  off ( x , y ) ,
γ = 1 MAR 1 ,
I c ( x , y ) = i = 1 N [ Mask i on ( x , y ) ] 2 I if ( x , y ) + MAR   i = 1 N Mask i on ( x , y ) I oof ( x , y ) ,
I nc ( x , y ) = i = 1 N Mask i on ( x , y ) [ 1 Mask i on ( x , y ) ] ×   I if ( x , y ) + MAR   i = 1 N [ 1 Mask i on ( x , y ) ] × I oof ( x , y ) .
ScaSub ( x , y ) = I if ( x , y ) = β [ I c ( x , y ) i = 1 N Mask i on ( x , y ) I nc ( x , y ) i = 1 N Mask i off ( x , y ) ] ,
β = N i = 1 N Mask i on ( x , y ) [ i = 1 N Mask i on ( x , y ) ] 2 N i = 1 N [ Mask i on ( x , y ) ] 2 [ i = 1 N Mask i on ( x , y ) ] 2 ,
ex i ( x ) = p i ( x ) h ex ( x ) .
p i ( x ) = j = 1 δ ( x s i j ) t ( x ) ,
E X i ( k ) = j = 1 [ α i j δ ( k S i j ) T ( k ) H ex ( k ) ] = j = 1 m [ α i j δ ( k S i j ) T ( k ) H ex ( k ) ] .
img i ( x ) = h em ( x ) [ obj ( x ) ex i ( x ) ] ,
IMG i ( k ) = H em [ OBJ   ( P i H ex ) ] = H em j = 1 m M ji sOBJ j ( k ) .
M j i = T j exp ( 2 π 1 α i k l + φ 0 ) .
α ij = α i k l = ( Δ x i k + Δ y i l ) ,
H e m { 1 + a | k | + b | k | 2 + c | k | 3 | k | < 1 0 | k | 1 ,

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