Abstract

Fluorescence emission difference microscopy (FED) obtains resolution-enhanced images by subtracting acquired solid and doughnut confocal images. Because of the mismatch of the outer contours of the two subtraction components, negative values are inevitable in the conventional FED method, giving rise to deformations. In this study, by using a saturation effect, we obtain imaging results with a profile-extended solid and center-shrunken doughnut point spread function. Owing to the nonlinear effect, two better-matched saturated images not only eliminate the deformations, but also enhance the resolving ability and signal to noise ratio compared to conventional FED. Simulations based on the saturated model of rhodamine 6G, as well as experiments on biological samples, are presented to verify the capability of the proposed concept, while experimental results show the unprecedented resolving ability of the saturated FED method.

© 2016 Optical Society of America

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References

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2016 (1)

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

2015 (8)

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

Y. Ma, C. Kuang, Y. Fang, B. Ge, D. Li, and X. Liu, “Virtual fluorescence emission difference microscopy based on photon reassignment,” Opt. Lett. 40(20), 4627–4630 (2015).
[Crossref] [PubMed]

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

N. Wang and T. Kobayashi, “Polarization modulation for fluorescence emission difference microscopy,” Opt. Express 23(10), 13704–13712 (2015).
[Crossref] [PubMed]

N. Wang and T. Kobayashi, “Numerical calibration of the spatial overlap for subtraction microscopy,” Opt. Express 23(10), 13410–13422 (2015).
[Crossref] [PubMed]

N. Tian, L. Fu, and M. Gu, “Resolution and contrast enhancement of subtractive second harmonic generation microscopy with a circularly polarized vortex beam,” Sci. Rep. 5, 13580 (2015).
[Crossref] [PubMed]

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241–275 (2015).
[Crossref]

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

2014 (5)

2013 (3)

2009 (1)

E. Rittweger, D. Wildanger, and S. Hell, “Far-field fluorescence nanoscopy of diamond color centers by ground state depletion,” Europhys. Lett. 86(1), 14001 (2009).
[Crossref]

2007 (2)

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

2006 (1)

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

2005 (2)

F. Gasparri, M. Mariani, F. Sola, and A. Galvani, “Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader,” Drug Discov. Today 9, 31–42 (2005).
[PubMed]

C. Eggeling, A. Volkmer, and C. A. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[Crossref] [PubMed]

2002 (1)

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]

1995 (2)

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

1994 (1)

1873 (1)

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv Für Mikroskopische Anatomie 9(1), 413–418 (1873).
[Crossref]

Abbe, E.

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv Für Mikroskopische Anatomie 9(1), 413–418 (1873).
[Crossref]

Antipov, A.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Baby, R.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Bianchini, P.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Carrington, W. A.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Côté, D.

Cremer, C.

Daradich, A.

De Koninck, Y.

Dehez, H.

Diaspro, A.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Ding, Z.

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

Eggeling, C.

C. Eggeling, A. Volkmer, and C. A. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[Crossref] [PubMed]

Eluru, G.

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241–275 (2015).
[Crossref]

Fang, Y.

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

Y. Ma, C. Kuang, Y. Fang, B. Ge, D. Li, and X. Liu, “Virtual fluorescence emission difference microscopy based on photon reassignment,” Opt. Lett. 40(20), 4627–4630 (2015).
[Crossref] [PubMed]

Y. Fang, Y. Wang, C. Kuang, and X. Liu, “Enhancing the resolution and contrast in CW-STED microscopy,” Opt. Commun. 322, 169–174 (2014).
[Crossref]

Fay, F. S.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Fogarty, K. E.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Fu, L.

N. Tian, L. Fu, and M. Gu, “Resolution and contrast enhancement of subtractive second harmonic generation microscopy with a circularly polarized vortex beam,” Sci. Rep. 5, 13580 (2015).
[Crossref] [PubMed]

Fujita, K.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

Galvani, A.

F. Gasparri, M. Mariani, F. Sola, and A. Galvani, “Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader,” Drug Discov. Today 9, 31–42 (2005).
[PubMed]

Gasecka, A.

Gasparri, F.

F. Gasparri, M. Mariani, F. Sola, and A. Galvani, “Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader,” Drug Discov. Today 9, 31–42 (2005).
[PubMed]

Ge, B.

Y. Ma, C. Kuang, Y. Fang, B. Ge, D. Li, and X. Liu, “Virtual fluorescence emission difference microscopy based on photon reassignment,” Opt. Lett. 40(20), 4627–4630 (2015).
[Crossref] [PubMed]

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

Ge, J.

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).
[Crossref] [PubMed]

Gorthi, S. S.

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241–275 (2015).
[Crossref]

Gu, M.

N. Tian, L. Fu, and M. Gu, “Resolution and contrast enhancement of subtractive second harmonic generation microscopy with a circularly polarized vortex beam,” Sci. Rep. 5, 13580 (2015).
[Crossref] [PubMed]

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).
[Crossref] [PubMed]

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]

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).
[Crossref] [PubMed]

Harke, B.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Heintzmann, R.

Hell, S.

E. Rittweger, D. Wildanger, and S. Hell, “Far-field fluorescence nanoscopy of diamond color centers by ground state depletion,” Europhys. Lett. 86(1), 14001 (2009).
[Crossref]

Hell, S. W.

Isenberg, G.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Jovin, T. M.

Kawano, S.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

Kawata, S.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

Kobayashi, M.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

Kobayashi, T.

Korobchevskaya, K.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Kozawa, Y.

Kuang, C.

Y. Ma, C. Kuang, Y. Fang, B. Ge, D. Li, and X. Liu, “Virtual fluorescence emission difference microscopy based on photon reassignment,” Opt. Lett. 40(20), 4627–4630 (2015).
[Crossref] [PubMed]

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

Y. Fang, Y. Wang, C. Kuang, and X. Liu, “Enhancing the resolution and contrast in CW-STED microscopy,” Opt. Commun. 322, 169–174 (2014).
[Crossref]

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(16), 1364–1371 (2014).
[Crossref]

S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (2014).
[Crossref] [PubMed]

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).
[Crossref] [PubMed]

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

Li, D.

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).
[Crossref] [PubMed]

Li, S.

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

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(16), 1364–1371 (2014).
[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).
[Crossref] [PubMed]

Li, Z.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Liu, S.

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

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).
[Crossref] [PubMed]

Liu, X.

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

Y. Ma, C. Kuang, Y. Fang, B. Ge, D. Li, and X. Liu, “Virtual fluorescence emission difference microscopy based on photon reassignment,” Opt. Lett. 40(20), 4627–4630 (2015).
[Crossref] [PubMed]

S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (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(16), 1364–1371 (2014).
[Crossref]

Y. Fang, Y. Wang, C. Kuang, and X. Liu, “Enhancing the resolution and contrast in CW-STED microscopy,” Opt. Commun. 322, 169–174 (2014).
[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).
[Crossref] [PubMed]

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

Lounis, B.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

Lynch, R. M.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Ma, Y.

Mariani, M.

F. Gasparri, M. Mariani, F. Sola, and A. Galvani, “Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader,” Drug Discov. Today 9, 31–42 (2005).
[PubMed]

Medda, R.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Moore, E. D.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Peres, C.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Piché, M.

Rittweger, E.

E. Rittweger, D. Wildanger, and S. Hell, “Far-field fluorescence nanoscopy of diamond color centers by ground state depletion,” Europhys. Lett. 86(1), 14001 (2009).
[Crossref]

Rong, Z.

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

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(16), 1364–1371 (2014).
[Crossref]

S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (2014).
[Crossref] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Sato, S.

Saxena, M.

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241–275 (2015).
[Crossref]

Segawa, S.

Seidel, C. A.

C. Eggeling, A. Volkmer, and C. A. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[Crossref] [PubMed]

Sheppard, C. J.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Sola, F.

F. Gasparri, M. Mariani, F. Sola, and A. Galvani, “Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader,” Drug Discov. Today 9, 31–42 (2005).
[PubMed]

Sun, S.

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

Tamarat, P.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

Tian, N.

N. Tian, L. Fu, and M. Gu, “Resolution and contrast enhancement of subtractive second harmonic generation microscopy with a circularly polarized vortex beam,” Sci. Rep. 5, 13580 (2015).
[Crossref] [PubMed]

Trebbia, J. B.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

Volkmer, A.

C. Eggeling, A. Volkmer, and C. A. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[Crossref] [PubMed]

Wang, N.

Wang, W.

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

Wang, Y.

Y. Fang, Y. Wang, C. Kuang, and X. Liu, “Enhancing the resolution and contrast in CW-STED microscopy,” Opt. Commun. 322, 169–174 (2014).
[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).
[Crossref] [PubMed]

Wichmann, J.

Wildanger, D.

E. Rittweger, D. Wildanger, and S. Hell, “Far-field fluorescence nanoscopy of diamond color centers by ground state depletion,” Europhys. Lett. 86(1), 14001 (2009).
[Crossref]

Willig, K. I.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Xu, Y.

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

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(16), 1364–1371 (2014).
[Crossref]

Yamanaka, M.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

Yang, B.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

You, S.

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (2014).
[Crossref] [PubMed]

Zhao, G.

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Adv. Opt. Photonics (1)

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241–275 (2015).
[Crossref]

AIP Adv. (1)

S. You, C. Kuang, S. Li, X. Liu, and Z. Ding, “Three-dimensional super-resolution imaging for fluorescence emission difference microscopy,” AIP Adv. 5(8), 084901 (2015).
[Crossref]

Archiv Für Mikroskopische Anatomie (1)

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv Für Mikroskopische Anatomie 9(1), 413–418 (1873).
[Crossref]

ChemPhysChem (1)

C. Eggeling, A. Volkmer, and C. A. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[Crossref] [PubMed]

Drug Discov. Today (1)

F. Gasparri, M. Mariani, F. Sola, and A. Galvani, “Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader,” Drug Discov. Today 9, 31–42 (2005).
[PubMed]

Europhys. Lett. (1)

E. Rittweger, D. Wildanger, and S. Hell, “Far-field fluorescence nanoscopy of diamond color centers by ground state depletion,” Europhys. Lett. 86(1), 14001 (2009).
[Crossref]

J. Microsc. (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]

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(16), 1364–1371 (2014).
[Crossref]

J. Opt. Soc. Am. A (1)

Nat. Methods (2)

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Nat. Photonics (1)

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, “Optical nanoscopy with excited state saturation at liquid helium temperatures,” Nat. Photonics 9(10), 658–662 (2015).
[Crossref]

Opt. Commun. (2)

Y. Fang, Y. Wang, C. Kuang, and X. Liu, “Enhancing the resolution and contrast in CW-STED microscopy,” Opt. Commun. 322, 169–174 (2014).
[Crossref]

Z. Rong, C. Kuang, Y. Fang, G. Zhao, Y. Xu, and X. Liu, “Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams,” Opt. Commun. 354, 71–78 (2015).
[Crossref]

Opt. Express (5)

Opt. Lett. (4)

Phys. Rev. Lett. (1)

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref] [PubMed]

Sci. Rep. (3)

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).
[Crossref] [PubMed]

N. Tian, L. Fu, and M. Gu, “Resolution and contrast enhancement of subtractive second harmonic generation microscopy with a circularly polarized vortex beam,” Sci. Rep. 5, 13580 (2015).
[Crossref] [PubMed]

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Science (2)

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[Crossref] [PubMed]

Other (2)

S. Liu, S. Sun, C. Kuang, B. Ge, W. Wang, and X. Liu, “Saturated virtual fluorescence emission difference microscopy based on detector array,” Opt. Commun. In press (2016).

G. Zhao, Z. Rong, C. Zheng, X. Liu, and C. Kuang, “3D fluorescence emission difference microscopy based on spatial light modulator,” J. Innovative Opt. Health Sci. (2016).

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Figures (10)

Fig. 1
Fig. 1 Saturation effect in fluorescence emission from rhodamine 6G molecules calculated using the five-level molecular electronic state model. The black dashed line denotes the excitation-emission curve with photo-bleaching ignored. The solid line denotes the excitation-emission curve with photo-bleaching considered. The blue line segment denotes the linear response portion, the red line segment denotes the nonlinear portion where the saturated effect is evident, and the green line segment denotes the portion where bleaching appears to be evident and cannot be ignored. The dashed black line represents the simulated data without any bleaching reaction.
Fig. 2
Fig. 2 Saturated PSFs of normalized intensity distributions at corresponding excitation intensities. Side lengths of each square are 2λ. (a)–(c) Confocal solid PSFs at 3 kW/cm2, 30 kW/cm2, and 100 kW/cm2, respectively. (d)–(f) Confocal doughnut PSFs at the corresponding irradiances. (g)–(i) FED PSFs at corresponding irradiances. The subtraction factors are all set to 0.72.
Fig. 3
Fig. 3 (a) Linear profiles of solid, doughnut, and FED results at the illumination intensity of 3 kW/cm2. (b) Linear profiles of the corresponding components at the illumination intensity of 30 kW/cm2. (c) Linear profiles of the corresponding components at the illumination intensity of 100 kW/cm2. The black, red, and green curves in (a)–(c) denote the linear profile of confocal, doughnut, and FED PSFs, respectively. (d) Linear profiles of confocal microscopy (black line, consistent to the ones in (a)-(c)), in conventional FED (red line, obtained without illumination intensity considered, with the subtraction factor of 0.72) and sFED (blue line, obtained from the red profile in (c)). (e) Normalized effective OTF profiles analogous to Fig. 3(d).
Fig. 4
Fig. 4 Flow chart of simulation process of sFED.
Fig. 5
Fig. 5 Imaging results of a 5 × 5-point array sample. (a) Designed array sample box, side lengths of the squares are 0.08λ, and the distance between adjacent points is 0.2λ. Normalized intensity imaging results with (b) confocal, (c) FED (r = 0.75), and (d) sFED (r = 1) with an excitation intensity of 100 kW/cm2.
Fig. 6
Fig. 6 Simulation results of a sample of computer simulated microtubules, side-lengths of the scope are 6λ. (a) Microtubules sample. Normalized intensity imaging results with (b) the confocal technique, (c) conventional FED technique (r = 0.75), and (d) sFED technique at the excitation intensity of 100 kW/cm2. (r = 0.8). (g)–(h) Magnified views of the regions indicated by the boxes in (a)–(d) with the corresponding color of the outer contour.
Fig. 7
Fig. 7 Schematic of the sFED system.
Fig. 8
Fig. 8 (a)–(d) Confocal results at the corresponding powers indicated on the top-right corner, (e)–(f) doughnut results at the corresponding powers indicated on the top-right corner, (i)–(l) comparison of an extracted confocal image, an FED image of a solid at illumination power of 4 µW subtracted by a doughnut at illumination power of 2.8 µW, a doughnut saturated FED image of a solid at illumination power of 4 µW subtracted by a doughnut at illumination power of 717 µW, and a saturated FED (sFED) image of a solid at illumination power of 128 µW subtracted by a doughnut at illumination power of 717 µW, respectively. The position of the single spot corresponds to the position indicated by the light blue box in (a). (m) Line profiles of confocal, FED, and sFED spots indicated by a green double arrow line in (i). Subtracted factors are set equally to 0.9 for a fair comparison. Pixel size is 15 nm, and the per dwell time is 0.1 ms.
Fig. 9
Fig. 9 Experimental results of 40 nm nanoparticles. (a)–(d) Sequentially acquired confocal images at an illumination power of 6.7 µW, doughnut images at an illumination power of 3.6 µW, saturated confocal images at an illumination power of 100 µW, and saturated doughnut images at an illumination power of 140 µW. (e) FED image obtained by subtracting (a) and (b). (f) sFED image obtained by subtracting (c) and (d). (g)–(h) Deconvoluted images of (e) and (f) with the Richardson-Lucy algorithm. (i1)–(i3) OTFs of confocal microscopy, FED, and sFED. (j1)–(j2) Histogram of FED, sFED. (k) Re-acquired saturated confocal image at an illumination power of 100 µW after the entire cycle of image acquisition from (a) to (d). (l) Line profiles of confocal, FED, sFED images indicated by the light blue line in (a). Notably, in order not to blur the corresponding spots in Figs. 9(e) and 9(f), positions of the normalized intensity profiles are only indicated by one light blue dashed line in Fig. 9 (a). Pixels size are 15 nm, and the per dwell time is 0.1 ms. The subtraction factors of both FED and sFED are set equally to 1 for a fair comparison.
Fig. 10
Fig. 10 Experimental results of vero cells. (a)–(d) Confocal image at the illumination power of 0.67 µW, FED image obtained by subtracting the confocal image at 0.6 µW and doughnut at 0.6 µW, doughnut saturated image obtained by subtracting the confocal image at 0.6 µW and saturated doughnut at 4 µW, sFED image obtained by subtracting the confocal image at 3 µW and doughnut at 4 µW, respectively. (e1)–(e4) Enlarged views of regions indicated by the white boxes in (a)–(d), (f1)–(f4) deconvoluted results of the magnified regions with an inserted colorbar for the above 2D figures. (g) Line profiles across the indicated line in the confocal, FED, and sFED images. Pixel size is 20 nm, and the per dwell time is 0.1 ms. All the subtraction factors are set equally to 0.89 for a fair comparison.

Equations (2)

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{ h c (x,y,z)= h em (x,y,z)( h det (x,y,z)p(x,y)) h em (x,y,z) h exc ξ(i) ,
I sFED = I ssolid r× I sdought ,

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