Abstract

The resolution of optical microscopy is limited by the numerical aperture and the wavelength of light. Many strategies for improving resolution such as 4Pi and I5M have focused on an increase of the numerical aperture. Other approaches have based resolution improvement in fluorescence microscopy on the establishment of a nonlinear relationship between local excitation light intensity in the sample and in the emitted light. However, despite their innovative character, current techniques such as stimulated emission depletion (STED) and ground-state depletion (GSD) microscopy require complex optical configurations and instrumentation to narrow the point-spread function. We develop the theory of nonlinear patterned excitation microscopy for achieving a substantial improvement in resolution by deliberate saturation of the fluorophore excited state. The postacquisition manipulation of the acquired data is computationally more complex than in STED or GSD, but the experimental requirements are simple. Simulations comparing saturated patterned excitation microscopy with linear patterned excitation microscopy (also referred to in the literature as structured illumination or harmonic excitation light microscopy) and ordinary widefield microscopy are presented and discussed. The effects of photon noise are included in the simulations.

© 2002 Optical Society of America

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    [CrossRef] [PubMed]
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2002 (1)

R. Heintzmann, C. Cremer, “Axial tomographic confocal fluorescence microscopy” J. Microsc. 206, 7–23 (2002).
[CrossRef] [PubMed]

2001 (4)

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

M. Nagorni, S. W. Hell, “Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. comparative study of concepts,” J. Opt. Soc. Am. A 18, 36–48 (2001).
[CrossRef]

B. Albrecht, A. V. Failla, R. Heintzmann, C. Cremer, “Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations,” J. Biomed. Opt. 6, 292–299 (2001).
[CrossRef] [PubMed]

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, 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]

2000 (4)

R. Heintzmann, G. Kreth, C. Cremer, “Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy—a method for improving 3D FISH images,” Anal. Cell Pathol. 20, 7–15 (2000).

J. T. Frohn, H. F. Knapp, 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]

T. A. Klar, S. Jakops, M. Dyba, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier brokenby stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
[CrossRef]

1999 (5)

A. Schönle, P. E. Hänninen, S. W. Hell, “Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy,” Ann. Phys. (Leipzig) 8, 115–133 (1999).
[CrossRef]

A. Schönle, S. W. Hell, “Far-field fluorescence microscopy with repetitive excitation,” Eur. Phys. J. D 6, 283–290 (1999).
[CrossRef]

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

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

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

1997 (3)

T. Wilson, R. Juskaitis, M. A. A. Neil, “A new approach to three dimensional imaging in microscopy,” Cell Vision 4, 231 (1997).

M. A. A. Neil, R. Juskaitis, T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905–1907 (1997).
[CrossRef]

S. W. Hell, M. Schrader, H. T. M. van der Voort, “Farfield fluorescence microscopy with three-dimensional resolution in the 100 nm range,” J. Microsc. 185, 1–5 (1997).
[CrossRef]

1996 (1)

R. Juskaitis, T. Wilson, M. A. A. Neil, M. Kozubek, “Efficient real-time confocal microscopy with white licht sources,” Nature (London) 383, 804–806 (1996).
[CrossRef]

1995 (1)

S. W. Hell, M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60, 495–497 (1995).
[CrossRef]

1994 (2)

S. W. Hell, J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19, 780–782 (1994).
[CrossRef] [PubMed]

S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[CrossRef]

1993 (2)

F. Lanni, B. Bailey, D. L. Farkas, D. L. Taylor, “Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,” Bioimaging 1, 187–196 (1993).
[CrossRef]

P. J. Sementilli, B. R. Hunt, M. S. Nadar, “Analysis of the limit to superresolution in incoherent imaging,” J. Opt. Soc. Am. A 10, 2265–2276 (1993).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

1989 (1)

P. J. Shaw, D. A. Agard, Y. Hirakoa, J. W. Sedat, “Tilted view reconstruction in optical microscopy: three dimensional reconstruction of drosophila melanogaster embryo nuclei,” Biophys. J. 55, 101–110 (1989).
[CrossRef] [PubMed]

1946 (1)

D. Gabor, “Theory of communication,” J. Inst. Electr. Eng. 63, 429–457 (1946).

Agard, D.

M. Gustafsson, J. Sedat, D. Agard, “Method and apparatus for three-dimensional microscopy with enhanced depth resolution,” U.S. patent5,671,085, September23, 1997.

Agard, D. A.

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

P. J. Shaw, D. A. Agard, Y. Hirakoa, J. W. Sedat, “Tilted view reconstruction in optical microscopy: three dimensional reconstruction of drosophila melanogaster embryo nuclei,” Biophys. J. 55, 101–110 (1989).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, J. W. Sedat, “3D widefield microscopy with two objective lenses: experimental verification of improved axial resolution,” in Three-Dimensional Microscopy: Image Acquisition and Processing III, C. J. Cogswell, G. S. Kino, T. Wilson, eds., Proc. SPIE2655, 62–66 (1996).
[CrossRef]

Albrecht, B.

B. Albrecht, A. V. Failla, R. Heintzmann, C. Cremer, “Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations,” J. Biomed. Opt. 6, 292–299 (2001).
[CrossRef] [PubMed]

Arndt-Jovin, D.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, 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, T. M. Jovin, “An optical sectioning programmable array microscope implemented with a digital micromirror device,” J. Microsc. 196, 317–331 (1999).
[CrossRef] [PubMed]

Bailey, B.

F. Lanni, B. Bailey, D. L. Farkas, D. L. Taylor, “Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,” Bioimaging 1, 187–196 (1993).
[CrossRef]

F. Lanni, D. L. Taylor, B. Bailey, “Field synthesis and optical subsectioning for standing wave microscopy,” U.S. patent5,801,881, September1, 1998.

Benedetti, P. A.

P. A. Benedetti, V. Evangelista, D. Guidarini, S. Vestri “Method for the acquisition of images by confocal,” U.S. patent6,016,367, January18, 2000.

Cremer, C.

R. Heintzmann, C. Cremer, “Axial tomographic confocal fluorescence microscopy” J. Microsc. 206, 7–23 (2002).
[CrossRef] [PubMed]

B. Albrecht, A. V. Failla, R. Heintzmann, C. Cremer, “Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations,” J. Biomed. Opt. 6, 292–299 (2001).
[CrossRef] [PubMed]

R. Heintzmann, G. Kreth, C. Cremer, “Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy—a method for improving 3D FISH images,” Anal. Cell Pathol. 20, 7–15 (2000).

S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[CrossRef]

R. Heintzmann, C. Cremer, “Lateral 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, P. M. Viallet, eds., Proc. SPIE3568, 185–196 (1999).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Dyba, M.

T. A. Klar, S. Jakops, M. Dyba, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier brokenby stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
[CrossRef]

Egner, A.

A. Egner, S. W. Hell, “Equivalence of the Huygens–Fresnel and Debye 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, S. Vestri “Method for the acquisition of images by confocal,” U.S. patent6,016,367, January18, 2000.

Failla, A. V.

B. Albrecht, A. V. Failla, R. Heintzmann, C. Cremer, “Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations,” J. Biomed. Opt. 6, 292–299 (2001).
[CrossRef] [PubMed]

Farkas, D. L.

F. Lanni, B. Bailey, D. L. Farkas, D. L. Taylor, “Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,” Bioimaging 1, 187–196 (1993).
[CrossRef]

Frohn, J. T.

J. T. Frohn, H. F. Knapp, 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, 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]

Gabor, D.

D. Gabor, “Theory of communication,” J. Inst. Electr. Eng. 63, 429–457 (1946).

Gemkov, M. J.

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, 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, S. Vestri “Method for the acquisition of images by confocal,” U.S. patent6,016,367, January18, 2000.

Gustafsson, M.

M. Gustafsson, J. Sedat, D. Agard, “Method and apparatus for three-dimensional microscopy with enhanced depth resolution,” U.S. patent5,671,085, September23, 1997.

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

M. G. L. Gustafsson, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII, J. Conchello, C. J. Cogswell, T. Wilson, eds., Proc. SPIE3919, 141–150 (2000).
[CrossRef]

M. G. L. Gustafsson, D. A. Agard, J. W. Sedat, “3D widefield microscopy with two objective lenses: experimental verification of improved axial resolution,” in Three-Dimensional Microscopy: Image Acquisition and Processing III, C. J. Cogswell, G. S. Kino, T. Wilson, eds., Proc. SPIE2655, 62–66 (1996).
[CrossRef]

Hanley, Q. S.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, 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, T. M. Jovin, “An optical sectioning programmable array microscope implemented with a digital micromirror device,” J. Microsc. 196, 317–331 (1999).
[CrossRef] [PubMed]

Hänninen, P.

P. Hänninen, S. Hell, “Luminescence-scanning microscopy process and a luminescence scanning microscope utilizing picosecond or greater pulse lasers,” U.S. patent5,777,732, July7, 1998.

Hänninen, P. E.

A. Schönle, P. E. Hänninen, S. W. Hell, “Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy,” Ann. Phys. (Leipzig) 8, 115–133 (1999).
[CrossRef]

Heintzmann, R.

R. Heintzmann, C. Cremer, “Axial tomographic confocal fluorescence microscopy” J. Microsc. 206, 7–23 (2002).
[CrossRef] [PubMed]

B. Albrecht, A. V. Failla, R. Heintzmann, C. Cremer, “Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations,” J. Biomed. Opt. 6, 292–299 (2001).
[CrossRef] [PubMed]

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, 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, G. Kreth, C. Cremer, “Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy—a method for improving 3D FISH images,” Anal. Cell Pathol. 20, 7–15 (2000).

R. Heintzmann, C. Cremer, “Lateral 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, P. M. Viallet, eds., Proc. SPIE3568, 185–196 (1999).
[CrossRef]

Hell, S.

P. Hänninen, S. Hell, “Luminescence-scanning microscopy process and a luminescence scanning microscope utilizing picosecond or greater pulse lasers,” U.S. patent5,777,732, July7, 1998.

Hell, S. W.

M. Nagorni, S. W. Hell, “Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. comparative study of concepts,” J. Opt. Soc. Am. A 18, 36–48 (2001).
[CrossRef]

T. A. Klar, S. Jakops, M. Dyba, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier brokenby stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
[CrossRef]

A. Schönle, P. E. Hänninen, S. W. Hell, “Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy,” Ann. Phys. (Leipzig) 8, 115–133 (1999).
[CrossRef]

A. Schönle, S. W. Hell, “Far-field fluorescence microscopy with repetitive excitation,” Eur. Phys. J. D 6, 283–290 (1999).
[CrossRef]

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

S. W. Hell, M. Schrader, H. T. M. van der Voort, “Farfield fluorescence microscopy with three-dimensional resolution in the 100 nm range,” J. Microsc. 185, 1–5 (1997).
[CrossRef]

S. W. Hell, M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60, 495–497 (1995).
[CrossRef]

S. W. Hell, J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19, 780–782 (1994).
[CrossRef] [PubMed]

S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[CrossRef]

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy: Nonlinear and Two-Photon-Induced Fluorescence, J. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, pp. 361–426.

Hirakoa, Y.

P. J. Shaw, D. A. Agard, Y. Hirakoa, J. W. Sedat, “Tilted view reconstruction in optical microscopy: three dimensional reconstruction of drosophila melanogaster embryo nuclei,” Biophys. J. 55, 101–110 (1989).
[CrossRef] [PubMed]

Hunt, B. R.

Ichie, K.

K. Ichie, “Laser scanning optical system and laser scanning optical apparatus,” U.S. patent5,796,112, August18, 1998.

Jakops, S.

T. A. Klar, S. Jakops, M. Dyba, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier brokenby stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
[CrossRef]

Jovin, T. M.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, 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, T. M. Jovin, “An optical sectioning programmable array microscope implemented with a digital micromirror device,” J. Microsc. 196, 317–331 (1999).
[CrossRef] [PubMed]

Juskaitis, R.

M. A. A. Neil, R. Juskaitis, T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905–1907 (1997).
[CrossRef]

T. Wilson, R. Juskaitis, M. A. A. Neil, “A new approach to three dimensional imaging in microscopy,” Cell Vision 4, 231 (1997).

R. Juskaitis, T. Wilson, M. A. A. Neil, M. Kozubek, “Efficient real-time confocal microscopy with white licht sources,” Nature (London) 383, 804–806 (1996).
[CrossRef]

T. Wilson, R. Juskaitis, M. A. A. Neil, M. Kozubeck, “An aperture correlation approach to confocal microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing IV, C. J. Cogswell, J. Conchello, T. Wilson, eds., Proc. SPIE2984, 21–23 (1997).
[CrossRef]

Klar, T. A.

T. A. Klar, S. Jakops, M. Dyba, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier brokenby stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
[CrossRef]

Knapp, H. F.

J. T. Frohn, H. F. Knapp, 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, 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]

Kozubeck, M.

T. Wilson, R. Juskaitis, M. A. A. Neil, M. Kozubeck, “An aperture correlation approach to confocal microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing IV, C. J. Cogswell, J. Conchello, T. Wilson, eds., Proc. SPIE2984, 21–23 (1997).
[CrossRef]

Kozubek, M.

R. Juskaitis, T. Wilson, M. A. A. Neil, M. Kozubek, “Efficient real-time confocal microscopy with white licht sources,” Nature (London) 383, 804–806 (1996).
[CrossRef]

Kreth, G.

R. Heintzmann, G. Kreth, C. Cremer, “Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy—a method for improving 3D FISH images,” Anal. Cell Pathol. 20, 7–15 (2000).

Kroug, M.

S. W. Hell, M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60, 495–497 (1995).
[CrossRef]

Lamb, W. E.

M. Sargent, M. O. Scully, W. E. Lamb, Laser Physics (Addison-Wesley, London, 1982) (4th printing).

Lanni, F.

F. Lanni, B. Bailey, D. L. Farkas, D. L. Taylor, “Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,” Bioimaging 1, 187–196 (1993).
[CrossRef]

F. Lanni, D. L. Taylor, B. Bailey, “Field synthesis and optical subsectioning for standing wave microscopy,” U.S. patent5,801,881, September1, 1998.

Lindek, S.

S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[CrossRef]

Minsky, M.

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467, December19, 1961.

Nadar, M. S.

Nagorni, M.

Neil, M. A. A.

T. Wilson, R. Juskaitis, M. A. A. Neil, “A new approach to three dimensional imaging in microscopy,” Cell Vision 4, 231 (1997).

M. A. A. Neil, R. Juskaitis, T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905–1907 (1997).
[CrossRef]

R. Juskaitis, T. Wilson, M. A. A. Neil, M. Kozubek, “Efficient real-time confocal microscopy with white licht sources,” Nature (London) 383, 804–806 (1996).
[CrossRef]

T. Wilson, R. Juskaitis, M. A. A. Neil, M. Kozubeck, “An aperture correlation approach to confocal microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing IV, C. J. Cogswell, J. Conchello, T. Wilson, eds., Proc. SPIE2984, 21–23 (1997).
[CrossRef]

Sandison, D. R.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 47–50.

Sargent, M.

M. Sargent, M. O. Scully, W. E. Lamb, Laser Physics (Addison-Wesley, London, 1982) (4th printing).

Schönle, A.

A. Schönle, S. W. Hell, “Far-field fluorescence microscopy with repetitive excitation,” Eur. Phys. J. D 6, 283–290 (1999).
[CrossRef]

A. Schönle, P. E. Hänninen, S. W. Hell, “Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy,” Ann. Phys. (Leipzig) 8, 115–133 (1999).
[CrossRef]

Schrader, M.

S. W. Hell, M. Schrader, H. T. M. van der Voort, “Farfield fluorescence microscopy with three-dimensional resolution in the 100 nm range,” J. Microsc. 185, 1–5 (1997).
[CrossRef]

Scully, M. O.

M. Sargent, M. O. Scully, W. E. Lamb, Laser Physics (Addison-Wesley, London, 1982) (4th printing).

Sedat, J.

M. Gustafsson, J. Sedat, D. Agard, “Method and apparatus for three-dimensional microscopy with enhanced depth resolution,” U.S. patent5,671,085, September23, 1997.

Sedat, J. W.

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

P. J. Shaw, D. A. Agard, Y. Hirakoa, J. W. Sedat, “Tilted view reconstruction in optical microscopy: three dimensional reconstruction of drosophila melanogaster embryo nuclei,” Biophys. J. 55, 101–110 (1989).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, J. W. Sedat, “3D widefield microscopy with two objective lenses: experimental verification of improved axial resolution,” in Three-Dimensional Microscopy: Image Acquisition and Processing III, C. J. Cogswell, G. S. Kino, T. Wilson, eds., Proc. SPIE2655, 62–66 (1996).
[CrossRef]

Sementilli, P. J.

Shaw, P. J.

P. J. Shaw, D. A. Agard, Y. Hirakoa, J. W. Sedat, “Tilted view reconstruction in optical microscopy: three dimensional reconstruction of drosophila melanogaster embryo nuclei,” Biophys. J. 55, 101–110 (1989).
[CrossRef] [PubMed]

Stelzer, E. H. K.

S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[CrossRef]

Stemmer, A.

J. T. Frohn, H. F. Knapp, 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, 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]

Strickler, J.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 47–50.

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Taylor, D. L.

F. Lanni, B. Bailey, D. L. Farkas, D. L. Taylor, “Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,” Bioimaging 1, 187–196 (1993).
[CrossRef]

F. Lanni, D. L. Taylor, B. Bailey, “Field synthesis and optical subsectioning for standing wave microscopy,” U.S. patent5,801,881, September1, 1998.

Tsien, R. Y.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 267–268.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 269, 272.

van der Voort, H. T. M.

S. W. Hell, M. Schrader, H. T. M. van der Voort, “Farfield fluorescence microscopy with three-dimensional resolution in the 100 nm range,” J. Microsc. 185, 1–5 (1997).
[CrossRef]

Verveer, P. J.

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

Vestri, S.

P. A. Benedetti, V. Evangelista, D. Guidarini, S. Vestri “Method for the acquisition of images by confocal,” U.S. patent6,016,367, January18, 2000.

Waggoner, A.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 269, 272.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 267–268.

Webb, W. W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 47–50.

Wells, K. S.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 47–50.

Wichmann, J.

Williams, R. M.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 47–50.

Wilson, T.

T. Wilson, R. Juskaitis, M. A. A. Neil, “A new approach to three dimensional imaging in microscopy,” Cell Vision 4, 231 (1997).

M. A. A. Neil, R. Juskaitis, T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905–1907 (1997).
[CrossRef]

R. Juskaitis, T. Wilson, M. A. A. Neil, M. Kozubek, “Efficient real-time confocal microscopy with white licht sources,” Nature (London) 383, 804–806 (1996).
[CrossRef]

T. Wilson, R. Juskaitis, M. A. A. Neil, M. Kozubeck, “An aperture correlation approach to confocal microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing IV, C. J. Cogswell, J. Conchello, T. Wilson, eds., Proc. SPIE2984, 21–23 (1997).
[CrossRef]

Anal. Cell Pathol. (1)

R. Heintzmann, G. Kreth, C. Cremer, “Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy—a method for improving 3D FISH images,” Anal. Cell Pathol. 20, 7–15 (2000).

Ann. Phys. (Leipzig) (1)

A. Schönle, P. E. Hänninen, S. W. Hell, “Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy,” Ann. Phys. (Leipzig) 8, 115–133 (1999).
[CrossRef]

Appl. Phys. B (1)

S. W. Hell, M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60, 495–497 (1995).
[CrossRef]

Appl. Phys. Lett. (1)

S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[CrossRef]

Bioimaging (1)

F. Lanni, B. Bailey, D. L. Farkas, D. L. Taylor, “Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,” Bioimaging 1, 187–196 (1993).
[CrossRef]

Biophys. J. (1)

P. J. Shaw, D. A. Agard, Y. Hirakoa, J. W. Sedat, “Tilted view reconstruction in optical microscopy: three dimensional reconstruction of drosophila melanogaster embryo nuclei,” Biophys. J. 55, 101–110 (1989).
[CrossRef] [PubMed]

Cell Vision (1)

T. Wilson, R. Juskaitis, M. A. A. Neil, “A new approach to three dimensional imaging in microscopy,” Cell Vision 4, 231 (1997).

Eur. Phys. J. D (1)

A. Schönle, S. W. Hell, “Far-field fluorescence microscopy with repetitive excitation,” Eur. Phys. J. D 6, 283–290 (1999).
[CrossRef]

J. Biomed. Opt. (1)

B. Albrecht, A. V. Failla, R. Heintzmann, C. Cremer, “Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations,” J. Biomed. Opt. 6, 292–299 (2001).
[CrossRef] [PubMed]

J. Inst. Electr. Eng. (1)

D. Gabor, “Theory of communication,” J. Inst. Electr. Eng. 63, 429–457 (1946).

J. Microsc. (7)

Q. S. Hanley, P. J. Verveer, M. J. Gemkov, D. Arndt-Jovin, 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, 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]

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

R. Heintzmann, C. Cremer, “Axial tomographic confocal fluorescence microscopy” J. Microsc. 206, 7–23 (2002).
[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]

S. W. Hell, M. Schrader, H. T. M. van der Voort, “Farfield fluorescence microscopy with three-dimensional resolution in the 100 nm range,” J. Microsc. 185, 1–5 (1997).
[CrossRef]

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

J. Opt. Soc. Am. A (2)

Nature (London) (1)

R. Juskaitis, T. Wilson, M. A. A. Neil, M. Kozubek, “Efficient real-time confocal microscopy with white licht sources,” Nature (London) 383, 804–806 (1996).
[CrossRef]

Opt. Lett. (3)

Proc. Natl. Acad. Sci. USA (2)

T. A. Klar, S. Jakops, M. Dyba, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier brokenby stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
[CrossRef]

J. T. Frohn, H. F. Knapp, 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]

Science (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Other (16)

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 269, 272.

P. A. Benedetti, V. Evangelista, D. Guidarini, S. Vestri “Method for the acquisition of images by confocal,” U.S. patent6,016,367, January18, 2000.

M. Sargent, M. O. Scully, W. E. Lamb, Laser Physics (Addison-Wesley, London, 1982) (4th printing).

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467, December19, 1961.

M. G. L. Gustafsson, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII, J. Conchello, C. J. Cogswell, T. Wilson, eds., Proc. SPIE3919, 141–150 (2000).
[CrossRef]

R. Heintzmann, C. Cremer, “Lateral 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, P. M. Viallet, eds., Proc. SPIE3568, 185–196 (1999).
[CrossRef]

T. Wilson, R. Juskaitis, M. A. A. Neil, M. Kozubeck, “An aperture correlation approach to confocal microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing IV, C. J. Cogswell, J. Conchello, T. Wilson, eds., Proc. SPIE2984, 21–23 (1997).
[CrossRef]

In the literature patterned excitation techniques have also been named structured illumination microscopy, harmonic excitation light microscopy (HELM) and laterally modulated excitation (LMEM).

M. G. L. Gustafsson, D. A. Agard, J. W. Sedat, “3D widefield microscopy with two objective lenses: experimental verification of improved axial resolution,” in Three-Dimensional Microscopy: Image Acquisition and Processing III, C. J. Cogswell, G. S. Kino, T. Wilson, eds., Proc. SPIE2655, 62–66 (1996).
[CrossRef]

M. Gustafsson, J. Sedat, D. Agard, “Method and apparatus for three-dimensional microscopy with enhanced depth resolution,” U.S. patent5,671,085, September23, 1997.

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy: Nonlinear and Two-Photon-Induced Fluorescence, J. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, pp. 361–426.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 47–50.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 267–268.

K. Ichie, “Laser scanning optical system and laser scanning optical apparatus,” U.S. patent5,796,112, August18, 1998.

P. Hänninen, S. Hell, “Luminescence-scanning microscopy process and a luminescence scanning microscope utilizing picosecond or greater pulse lasers,” U.S. patent5,777,732, July7, 1998.

F. Lanni, D. L. Taylor, B. Bailey, “Field synthesis and optical subsectioning for standing wave microscopy,” U.S. patent5,801,881, September1, 1998.

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Figures (7)

Fig. 1
Fig. 1

Fundamental concept of SPEM implemented by fluorophore saturation. (a) Pattern of the sinusoidal emittability distribution generated by low-intensity patterned excitation (linear patterned excitation microscopy). (b) Scheme of the emittability pattern of (a) in Fourier space. The vertical arrows denote the frequency maxima corresponding to the sinusoidal pattern of the excitation. The structure of the Fourier-transformed object density distribution ρ+1˜(k) attached to one of those maxima is also shown. Other such components are omitted for clarity. (c) Emittability distribution in real space with high-intensity illumination leading to fluorophore saturation. This emittability distribution was used in the SPEM simulations. (d) Corresponding emittability pattern in Fourier space. The arrows denote the maxima, which are caused by the nonlinear distortion (by fluorophore saturation) of the sinusoidal intensity pattern (a), (b). The Fourier-transformed object distribution is attached to every maximum [for clarity this is shown only for the second maximum ρ+2˜(k)].

Fig. 2
Fig. 2

Possible realizations of the SPEM concept by far-field epi-fluorescence microscopy. (a) The SLM could be a diffraction pattern generated by a mask, an LCD, or a digital mirror device. To introduce the nonlinearity, a very bright light source is needed. (b) Implementation with a coherent laser source for the generation of standing waves. (c) Improved version of (a) with use of a phase-modulating SLM to permit a high degree of modulation as well as a high light efficiency on the illumination side.

Fig. 3
Fig. 3

Simulations of resolution enhancement in SPEM. (a) The object, displayed with a slight clipping at high intensities. The object was simulated to contain a background fluorescence level (resulting in ∼7000 expected photons per pixel in its image) so that the grating pattern can be observed in the images. (b)–(f) Virtual microscopic images simulated with a theoretic PSF and added Poisson noise (maximum pixel set to 104 expected photons). (b) Simulated image under linear patterned excitation conditions (phase 1). (c), (d) Images simulated at phase 1 and phase 4 of the illumination pattern (out of 7 phases) with a relative saturation of α=5/6. (e), (f) Other images simulated at different directions of patterning.

Fig. 4
Fig. 4

High-resolution reconstructions from the images shown in Fig. 3 and with other directions of the patterned illumination. (a) Convolution of the object [Fig. 3(a)] with the widefield PSF and application of photon noise corresponding to 104 expected photons at the maximum. (b) Application of a high-frequency enhancement (γ=2%), Eq. (10), to the image shown in (a). A simulation with a maximum of 106 photons yielded a much clearer image (data not shown). (c) Reconstruction from simulated images with use of four directions of patterned illumination at nonsaturating excitation intensities, accounting for m=±1 orders in the reconstruction. High-frequency enhancement was applied. (d) Reconstruction of SPEM data [α=5/6, Eq. (11)] accounting for m=±3 orders with a successive application of high-frequency enhancement (γ=2%), Eq. (10).

Fig. 5
Fig. 5

Effective PSFs corresponding to different simulations. (a) Widefield PSF without filtering. (b) The result after filtering. (c) PSF including the reconstruction process in the linear case with four patterning directions and m=±1 orders including high-frequency enhancement. (d) PSF with use of SPEM with four patterning directions and m=±3 orders and successive filtering. (e), (f) Respective OTFs of (c) and (d).

Fig. 6
Fig. 6

One-dimensional cuts through the respective simulated (a), (b) PSFs or (c), (d) OTFs. (b), (d) With high-frequency enhancement (γ=2%) and (a), (c) without high-frequency enhancement.

Fig. 7
Fig. 7

Simulated application of the SPEM concept to a slice of a cell nucleus. (a) Intensity-inverted part of an electron micrograph of an embryonal bovine cell nucleus near its nuclear membrane displaying the “nuclear matrix.” Image (a) has been used for SPEM simulations in which every image was adjusted to a maximum of 104 expected photons. (b) Simulated fluorescence widefield image with successive high-frequency filtering (γ=2%). (c) Reconstruction using linear patterned excitation microscopy (104 photons in maximum, m=±1 orders) including high-frequency enhancement (γ=2%). (d) SPEM reconstruction with high-frequency enhancement (γ=2%).

Equations (16)

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

I(x)=hem(x)(Iex(x)ρ(x)).
I˜(k)=hem˜(k)(Iex˜(k)ρ˜(k)).
hem(x)Iem(ρ(x), Iex(x)),
hem˜(k)Iem˜(ρ(k), Iex(k)),
Iem(x)c0+c1ρ(x)+c2Iex(x)+c3ρ(x)Iex(x)+c4ρ(x)Iex(x)2+c5ρ(x)Iex(x)3+ .
Iem(x)ρ(x)[c1+c3Iex(x)+c4Iex(x)2+c5Iex(x)3+],
Iem(x)=ρ(x)Em(x),
Em(x)=i=1ciIex(x)i.
Iem˜(k)=ρ˜(k)Em˜(k)
IemkfΨex1στ+Ψex,
Ihfe˜=Irec˜1|Prec˜|+γ.
α=Ψex1στ+Ψex.
hem˜(k)[ϕlδ(k-Δkl)ρ˜(k)],
In˜(k)=l=-mmMlnρ˜l(k),
Mln=cl exp(2πi ln/s),
l{-mm},n{0s-1}.

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