Abstract

We propose a method for eliminating the deformations in fluorescence emission difference microscopy (FED). Due to excessive subtraction, negative values are inevitable in the original FED method, giving rise to deformations. We propose modulating the beam to generate an extended solid focal spot and a hollow focal spot. Negative image values can be avoided by using these two types of excitation spots in FED imaging. Hence, deformations are eliminated, and the signal-to-noise ratio is improved. In deformation-free imaging, the resolution is higher than that of confocal imaging by 32%. Compared to standard FED imaging with the same level of deformations, our method provides superior resolution.

© 2014 Optical Society of America

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References

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  1. T. Wilson, Confocal Microscopy (Academic, 1990), Vol. 426, pp. 1–64.
  2. D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).
  3. J. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2010).
  4. 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(5793), 1642–1645 (2006).
    [Crossref] [PubMed]
  5. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
    [Crossref] [PubMed]
  6. M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
    [Crossref] [PubMed]
  7. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [Crossref] [PubMed]
  8. T. Dertinger, R. Colyer, R. Vogel, M. Heilemann, M. Sauer, J. Enderlein, and S. Weiss, “Superresolution optical fluctuation imaging (SOFI),” in Nano-Biotechnology for Biomedical and Diagnostic Research (Springer, 2012), pp. 17–21.
  9. R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
    [Crossref] [PubMed]
  10. Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
    [Crossref]
  11. C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
  12. S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
    [Crossref]
  13. Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).
  14. H. Dehez, M. Piché, and Y. De Koninck, “Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging,” Opt. Express 21(13), 15912–15925 (2013).
    [Crossref] [PubMed]
  15. H. Dehez, M. Piché, and Y. De Koninck, “High resolution imaging with TM01 laser beams,” in Photonics North 2009 (International Society for Optics and Photonics, 2009), 738606.
  16. S. Segawa, Y. Kozawa, and S. Sato, “Resolution enhancement of confocal microscopy by subtraction method with vector beams,” Opt. Lett. 39(11), 3118–3121 (2014).
    [Crossref] [PubMed]
  17. G. Vicidomini, R. Schmidt, A. Egner, S. Hell, and A. Schönle, “Automatic deconvolution in 4Pi-microscopy with variable phase,” Opt. Express 18(10), 10154–10167 (2010).
    [Crossref] [PubMed]
  18. J. Bewersdorf, R. Schmidt, and S. W. Hell, “Comparison of I5M and 4Pi-microscopy,” J. Microsc. 222(2), 105–117 (2006).
    [Crossref] [PubMed]
  19. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
    [Crossref]
  20. X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
    [Crossref]
  21. M. Muller, Introduction to Confocal Fluorescence Microscopy (SPIE, 2006), Chap. 1.

2014 (2)

S. Segawa, Y. Kozawa, and S. Sato, “Resolution enhancement of confocal microscopy by subtraction method with vector beams,” Opt. Lett. 39(11), 3118–3121 (2014).
[Crossref] [PubMed]

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

2013 (4)

H. Dehez, M. Piché, and Y. De Koninck, “Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging,” Opt. Express 21(13), 15912–15925 (2013).
[Crossref] [PubMed]

Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
[Crossref]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

2010 (2)

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

G. Vicidomini, R. Schmidt, A. Egner, S. Hell, and A. Schönle, “Automatic deconvolution in 4Pi-microscopy with variable phase,” Opt. Express 18(10), 10154–10167 (2010).
[Crossref] [PubMed]

2006 (3)

J. Bewersdorf, R. Schmidt, and S. W. Hell, “Comparison of I5M and 4Pi-microscopy,” J. Microsc. 222(2), 105–117 (2006).
[Crossref] [PubMed]

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

2003 (1)

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

2000 (1)

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

1994 (1)

1989 (1)

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

1959 (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Bewersdorf, J.

J. Bewersdorf, R. Schmidt, and S. W. Hell, “Comparison of I5M and 4Pi-microscopy,” J. Microsc. 222(2), 105–117 (2006).
[Crossref] [PubMed]

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

De Koninck, Y.

Dehez, H.

Egner, A.

Ge, J.

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Gu, Z.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
[Crossref]

Gustafsson, M. G.

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

Hanley, Q. S.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

Hao, X.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Heintzmann, R.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

Hell, S.

Hell, S. W.

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Jovin, T. M.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

Kozawa, Y.

Kuang, C.

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
[Crossref]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Li, H.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Li, S.

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

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(5793), 1642–1645 (2006).
[Crossref] [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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Liu, W.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Liu, X.

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
[Crossref]

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Munroe, P.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

Nailon, J.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

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(5793), 1642–1645 (2006).
[Crossref] [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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Piché, M.

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Rong, Z.

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Sarafis, V.

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

Sato, S.

Schmidt, R.

Schönle, A.

Segawa, S.

Shotton, D. M.

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Vicidomini, G.

Wang, T.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Wang, Y.

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
[Crossref]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Wichmann, J.

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Xu, Y.

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

J. Cell Sci. (1)

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

J. Microsc. (2)

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

J. Bewersdorf, R. Schmidt, and S. W. Hell, “Comparison of I5M and 4Pi-microscopy,” J. Microsc. 222(2), 105–117 (2006).
[Crossref] [PubMed]

J. Mod. Opt. (1)

Z. Rong, S. Li, C. Kuang, Y. Xu, and X. Liu, “Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy,” J. Mod. Opt. 61, 1364–1371 (2014).

J. Opt. (2)

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15(12), 125708 (2013).
[Crossref]

Micron (1)

R. Heintzmann, V. Sarafis, P. Munroe, J. Nailon, Q. S. Hanley, and T. M. Jovin, “Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes,” Micron 34(6–7), 293–300 (2003).
[Crossref] [PubMed]

Nat. Methods (1)

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Opt. Express (2)

Opt. Laser Technol. (1)

Y. Wang, C. Kuang, Z. Gu, and X. Liu, “Image subtraction method for improving lateral resolution and SNR in confocal microscopy,” Opt. Laser Technol. 48, 489–494 (2013).
[Crossref]

Opt. Lett. (2)

Proc. R. Soc. Lond. A Math. Phys. Sci. (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Sci. Rep. (1)

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).

Science (1)

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(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Other (5)

T. Wilson, Confocal Microscopy (Academic, 1990), Vol. 426, pp. 1–64.

J. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2010).

T. Dertinger, R. Colyer, R. Vogel, M. Heilemann, M. Sauer, J. Enderlein, and S. Weiss, “Superresolution optical fluctuation imaging (SOFI),” in Nano-Biotechnology for Biomedical and Diagnostic Research (Springer, 2012), pp. 17–21.

M. Muller, Introduction to Confocal Fluorescence Microscopy (SPIE, 2006), Chap. 1.

H. Dehez, M. Piché, and Y. De Koninck, “High resolution imaging with TM01 laser beams,” in Photonics North 2009 (International Society for Optics and Photonics, 2009), 738606.

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Figures (8)

Fig. 1
Fig. 1 FED system setup: PBS, polarizing beam splitter; PP1 and PP2, phase plate; D, diaphragm; S1 and S2, shutter; S, sample; M, reflecting mirror; DM, dichroic mirror; QWP, quarter-wave plate; OL, objective lens; CL, converging lens; and PH, pinhole.
Fig. 2
Fig. 2 Focal spot pattern under different modulations. (a) Normal focal spot. (b) Hollow focal spot. (c) Extended solid focal spot. (d) Further extended solid focal spot. The curve beneath each pattern is the linear profile of the intensity distribution along the central axis.
Fig. 3
Fig. 3 Point spread functions associated with the confocal imaging resulting from illumination with different focal spot. (a) PSFs resulting from illumination with a normal solid spot and with a hollow spot. (b) PSFs resulting from illumination with an extended solid spot and with a hollow spot. (c) PSFs resulting from illumination with a further extended solid spot and with a hollow spot. The shaded area shows where a negative PSF difference may occur.
Fig. 4
Fig. 4 Point spread functions for FED imaging. (a) PSF of the original FED method. The subtractive factor is 0.4. (b) PSF of deformation-free FED imaging. The subtractive factor is 0.95. (c) and (d) are respectively the linear profiles of (a) and (b) along the central axis.
Fig. 5
Fig. 5 (a) Minimum value of PSFs under different subtractive factors. (b) FWHMs of PSFs under different subtractive factors. (c) Relation between minimum value and PSF FWHM.
Fig. 6
Fig. 6 Simulation results for imaging a sample array pattern consisting of nine points. (a) Actual array pattern, consisting of points of unequal intensities, (b) confocal image, (c) dfFED image, where the subtractive factor is 0.95, and (d)–(f) FED images, with subtractive factors of 0.4, 0.6, and 0.8 respectively.
Fig. 7
Fig. 7 Simulation results for imaging a sample array pattern of parallel lines. (a) Original sample pattern, (b) dfFED image, where the subtractive factor is 1.00, (c) FED image, where the subtractive factor is 0.60, and (d) linear intensity profiles, taken along horizontal lines through the centers of (b) and (c).
Fig. 8
Fig. 8 Simulation results for imaging a spoke-like sample pattern. (a) Spoke-like sample pattern, (b) FED image, where the subtractive factor is 0.51, (c) dfFED image, where the subtractive factor is 1.029, and (d) linear profiles of intensity along the green lines in (a), (b), and (c). The minimum values in (b) and (c) are both −0.2.

Equations (5)

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I FED = I solid r× I hollow
PS F c =PS F e ×(PS F f p)
PS F FED =PS F c1 r×PS F c2
PS F FED =PS F normal r×PS F hollow
PS F dfFED =PS F extended+ r×PS F hollow

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