Abstract

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

© 2012 OSA

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]

2012

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Y.-T. Kao, X. Zhu, and W. Min, “Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein,” Proc. Natl. Acad. Sci. U.S.A.109(9), 3220–3225 (2012).
[CrossRef] [PubMed]

L. Wei, Z. Chen, and W. Min, “Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging,” Biomed. Opt. Express3(6), 1465–1475 (2012).
[CrossRef]

2011

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
[CrossRef] [PubMed]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011).
[CrossRef] [PubMed]

N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011).
[CrossRef] [PubMed]

2010

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent proteins and their applications in imaging living cells and tissues,” Physiol. Rev.90(3), 1103–1163 (2010).
[CrossRef] [PubMed]

K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express1(3), 791–797 (2010).
[CrossRef] [PubMed]

F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
[CrossRef] [PubMed]

2009

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express17(16), 13354–13364 (2009).
[CrossRef] [PubMed]

M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009).
[CrossRef]

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.19(11), 555–565 (2009).
[CrossRef] [PubMed]

2008

J. N. D. Kerr and W. Denk, “Imaging in vivo: watching the brain in action,” Nat. Rev. Neurosci.9(3), 195–205 (2008).
[CrossRef] [PubMed]

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

N. Chen, C.-H. Wong, and C. J. Sheppard, “Focal modulation microscopy,” Opt. Express16(23), 18764–18769 (2008).
[CrossRef] [PubMed]

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

2007

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

2006

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A23(12), 3139–3149 (2006).
[CrossRef] [PubMed]

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

2005

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A.102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

2004

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003).
[CrossRef] [PubMed]

1999

1990

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Aaron, H. L.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Alfano, R. R.

Ando, R.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Andresen, M.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Benninger, R. K. P.

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Ben-Yakar, A.

N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011).
[CrossRef] [PubMed]

Betzig, E.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

Bock, H.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

Chen, N.

Chen, Z.

Chudakov, D. M.

D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent proteins and their applications in imaging living cells and tissues,” Physiol. Rev.90(3), 1103–1163 (2010).
[CrossRef] [PubMed]

Davidson, M. W.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Dedecker, P.

M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009).
[CrossRef]

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

Denk, W.

J. N. D. Kerr and W. Denk, “Imaging in vivo: watching the brain in action,” Nat. Rev. Neurosci.9(3), 195–205 (2008).
[CrossRef] [PubMed]

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A23(12), 3139–3149 (2006).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Drobizhev, M.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

Durr, N. J.

N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011).
[CrossRef] [PubMed]

Durst, M. E.

Easley, C. J.

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Eggeling, C.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
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M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A.102(49), 17565–17569 (2005).
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M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
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Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008).
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C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
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R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
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M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
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H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
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F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
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G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
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F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
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E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
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H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
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Hashimoto, H.

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M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009).
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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A.102(49), 17565–17569 (2005).
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F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
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M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009).
[CrossRef]

R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
[CrossRef] [PubMed]

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

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M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A.102(49), 17565–17569 (2005).
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N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011).
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D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011).
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C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

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M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

Isacoff, E. Y.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Isobe, K.

Jackson, D.

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Jackson, D. K.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A.102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

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E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

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E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Kannari, F.

Kao, Y.-T.

Y.-T. Kao, X. Zhu, and W. Min, “Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein,” Proc. Natl. Acad. Sci. U.S.A.109(9), 3220–3225 (2012).
[CrossRef] [PubMed]

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H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express1(3), 791–797 (2010).
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Kurokawa, H.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

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A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

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A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
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B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
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J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.19(11), 555–565 (2009).
[CrossRef] [PubMed]

Liu, F.

Lukyanov, K. A.

F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
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D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent proteins and their applications in imaging living cells and tissues,” Physiol. Rev.90(3), 1103–1163 (2010).
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D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent proteins and their applications in imaging living cells and tissues,” Physiol. Rev.90(3), 1103–1163 (2010).
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M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006).
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E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Makarov, N. S.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

Mao, S.

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Marriott, G.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Matz, M. V.

D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent proteins and their applications in imaging living cells and tissues,” Physiol. Rev.90(3), 1103–1163 (2010).
[CrossRef] [PubMed]

Mertz, J.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

Midorikawa, K.

Milkie, D. E.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

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L. Wei, Z. Chen, and W. Min, “Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging,” Biomed. Opt. Express3(6), 1465–1475 (2012).
[CrossRef]

Y.-T. Kao, X. Zhu, and W. Min, “Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein,” Proc. Natl. Acad. Sci. U.S.A.109(9), 3220–3225 (2012).
[CrossRef] [PubMed]

Miyawaki, A.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express1(3), 791–797 (2010).
[CrossRef] [PubMed]

R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
[CrossRef] [PubMed]

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Mizuno, H.

K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express1(3), 791–797 (2010).
[CrossRef] [PubMed]

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Nishimura, N.

Noda, H.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

Patterson, G. H.

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.19(11), 555–565 (2009).
[CrossRef] [PubMed]

Petchprayoon, C.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Piatkevich, K. D.

B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
[CrossRef] [PubMed]

Piston, D. W.

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Psaltis, D.

Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008).
[CrossRef] [PubMed]

Ran, J.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Rebane, A.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

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E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Rueckel, M.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

Sakata, T.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Sakaue-Sawano, A.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

Sauer, M.

M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009).
[CrossRef]

Schaffer, C. B.

Schönle, A.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

Shao, L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Sheppard, C. J.

Shimogori, T.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

Singer, R. H.

B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
[CrossRef] [PubMed]

Stiel, A. C.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Subach, F. V.

F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
[CrossRef] [PubMed]

Suda, A.

Testa, I.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

Theer, P.

Tillo, S. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

Trowitzsch, S.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Tulyathan, O.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Uji-i, H.

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

Urban, N. T.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

Verkhusha, V. V.

B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
[CrossRef] [PubMed]

F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
[CrossRef] [PubMed]

Wahl, M. C.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Warp, E.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Weber, G.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Wei, L.

Weisspfennig, C. T.

N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011).
[CrossRef] [PubMed]

Wenzel, D.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Willig, K. I.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

Winoto, L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

Wong, A. W.

Wong, C.-H.

Wu, B.

B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
[CrossRef] [PubMed]

Xu, C.

Yan, Y.

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
[CrossRef] [PubMed]

Yang, C.

Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008).
[CrossRef] [PubMed]

Yaqoob, Z.

Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008).
[CrossRef] [PubMed]

Ying, J.

Zhang, L.

F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
[CrossRef] [PubMed]

Zhu, X.

Y.-T. Kao, X. Zhu, and W. Min, “Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein,” Proc. Natl. Acad. Sci. U.S.A.109(9), 3220–3225 (2012).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Appl. Opt.

Biomed. Opt. Express

Biophys. J.

S. Mao, R. K. P. Benninger, Y. Yan, C. Petchprayoon, D. Jackson, C. J. Easley, D. W. Piston, and G. Marriott, “Optical lock-in detection of FRET using synthetic and genetically encoded optical switches,” Biophys. J.94(11), 4515–4524 (2008).
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R. Ando, C. Flors, H. Mizuno, J. Hofkens, and A. Miyawaki, “Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants,” Biophys. J.92(12), L97–L99 (2007).
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A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J.94(4), 1449–1458 (2008).
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Chem. Biol.

F. V. Subach, L. Zhang, T. W. J. Gadella, N. G. Gurskaya, K. A. Lukyanov, and V. V. Verkhusha, “Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET,” Chem. Biol.17(7), 745–755 (2010).
[CrossRef] [PubMed]

Curr. Opin. Cell Biol.

B. Wu, K. D. Piatkevich, T. Lionnet, R. H. Singer, and V. V. Verkhusha, “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics,” Curr. Opin. Cell Biol.23(3), 310–317 (2011).
[CrossRef] [PubMed]

J. Am. Chem. Soc.

C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc.129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

J. Biomed. Opt.

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011).
[CrossRef] [PubMed]

N. J. Durr, C. T. Weisspfennig, B. A. Holfeld, and A. Ben-Yakar, “Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues,” J. Biomed. Opt.16(2), 026008 (2011).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Laser Photon. Rev.

M. Heilemann, P. Dedecker, J. Hofkens, and M. Sauer, “Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification,” Laser Photon. Rev.3(1-2), 180–202 (2009).
[CrossRef]

Nat. Biotechnol.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy,” Nat. Biotechnol.26(9), 1035–1040 (2008).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Methods

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

Nat. Neurosci.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci.14(11), 1481–1488 (2011).
[CrossRef] [PubMed]

Nat. Photonics

Z. Yaqoob, D. Psaltis, M. S. Feld, and C. Yang, “Optical phase conjugation for turbidity suppression in biological samples,” Nat. Photonics2(2), 110–115 (2008).
[CrossRef] [PubMed]

Nat. Rev. Neurosci.

J. N. D. Kerr and W. Denk, “Imaging in vivo: watching the brain in action,” Nat. Rev. Neurosci.9(3), 195–205 (2008).
[CrossRef] [PubMed]

Nature

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478(7368), 204–208 (2011).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Physiol. Rev.

D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent proteins and their applications in imaging living cells and tissues,” Physiol. Rev.90(3), 1103–1163 (2010).
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Proc. Natl. Acad. Sci. U.S.A.

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A.102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012).
[CrossRef] [PubMed]

G. Marriott, S. Mao, T. Sakata, J. Ran, D. K. Jackson, C. Petchprayoon, T. J. Gomez, E. Warp, O. Tulyathan, H. L. Aaron, E. Y. Isacoff, and Y. Yan, “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells,” Proc. Natl. Acad. Sci. U.S.A.105(46), 17789–17794 (2008).
[CrossRef] [PubMed]

Y.-T. Kao, X. Zhu, and W. Min, “Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein,” Proc. Natl. Acad. Sci. U.S.A.109(9), 3220–3225 (2012).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A.104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A.103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

Science

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science306(5700), 1370–1373 (2004).
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J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.19(11), 555–565 (2009).
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Other

B. R. Masters and P. T. C. So, eds., Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008).

R. Yuste, ed., Imaging: A Laboratory Manual (Cold Spring Harbor Press, 2010).

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Figures (5)

Fig. 1
Fig. 1

Fundamental imaging-depth limit of multi-photon fluorescence microscopy. (A) Depth-dependent two-photon optical sections of a tissue phantom made of 5% intralipid, 2% agarose gel and fluorescent beads (diameter 0.9 µm) under a constant laser power excitation. Fluorescence signal quickly attenuates with the imaging depth. (B) Depth images of the same sample using a compensative higher laser power to maintain the signal strength. The resulting images can reach deeper than (A), but their contrast deteriorates as the out-of-focus background begins to dominate. The fundamental imaging-depth limit is defined when the in-focus signal and the out-of-focus background are equal to each other.

Fig. 2
Fig. 2

One-photon and two-photon induced photoswitching of Dronpa-3 protein. (A) Upon irradiation at 405 and 488 nm, Dronpa-3 switches between dark and bright states in a reversible manner (cyan and purple dashed lines), where the downward black arrow and green arrow indicates the non-radiative relaxation from the excited dark state and florescence decay from the excited bright state, respectively. (B) Time-lapse (in seconds) two-photon images (at 920nm) of Dronpa-3 expressing E. coli cells undergoing bright-to-dark switching upon the same 920nm irradiation. A two-times higher 920nm laser power leads to notably faster switching-off kinetics (lower panel). (C) Time-lapse (in seconds) two-photon images (at 920nm) of Dronpa-3 expressing E. coli cells undergoing dark-to-bright switching upon 800nm irradiation (applied between adjacent images). A two-times higher 800nm laser power leads to notably faster switching-on kinetics (lower panel).

Fig. 3
Fig. 3

Principles of multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI) with RSFPs. (A) MPDI. For the regular pre-switching image, when imaging deep into the scattering sample, substantial laser intensity is distributed out of focus, generating background that is comparable to the in-focus signal. In the post-switching image, in-focus RSFPs are switched off much more than those out of focus, creating a disparity of dark-bright states in space. The resulting difference image leads to significantly improved contrast. (B) MPAI. RSFPs which are originally in the bright state will be completely switched off into the dark state. The subsequent multiphoton activation will switch on a higher percentage of RSFPs at focus than those out of focus. This spatial disparity of dark-bright transitions leads to a significantly decreased background in the final multiphoton imaging step.

Fig. 4
Fig. 4

Experimental demonstration of multiphoton deactivation and imaging (MPDI) on tissue phantoms. (A) For Dronpa-3 expressing E. coli cells packed in 3D, the regular pre-switching image (at 920nm) is overwhelming at a depth of 250 µm. After performing a slow deactivation scanning, the post-deactivation image is dimmer. The difference image (after auto-scaled) offers a much improved image contrast. (B) Similar contrast improvement is observed for HEK 293T cells (transfected by H2B-Dronpa plasmids) placed on a dense layer of scattering E. coli cells expressing Dronpa-3.

Fig. 5
Fig. 5

Experimental demonstration of multiphoton activation and imaging (MPAI) on tissue phantoms. (A) For Dronpa-3 expressing E. coli cells mixed with polystyrene beads packed in 3D, the background in the bright-state image (at 920nm) is overwhelming at a depth of 140µm. A brief 488nm laser illumination converted Dronpa-3 in the volume of interest completely to the dark state. Afterwards, a relatively weak 800nm pulsed laser was used to activate Dronpa-3 prior to the subsequent two-photon imaging at 920nm. The resulting MPAI image reveals features that are buried in the original image of the bright state. (B) Similar contrast improvement is observed for HEK 293T cells (transfected by H2B-Dronpa-3 plasmids) placed on a 120µm-thick layer of Dronpa-3 expressing E. coli cells.

Equations (1)

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( S B ) regular = V in 0 τ C S (r,z) I 2 (r,z,t)dtdV V out 0 τ C B (r,z) I 2 (r,z,t)dtdV =1

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