Abstract

It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

© 2012 OSA

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2012 (1)

2011 (4)

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

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]

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]

2010 (2)

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]

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

2009 (3)

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]

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]

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]

2008 (2)

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]

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008).
[CrossRef] [PubMed]

2007 (1)

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

2006 (5)

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]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

2005 (2)

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

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[CrossRef] [PubMed]

2004 (2)

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[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]

2003 (2)

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

2002 (1)

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

1999 (1)

1998 (2)

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A.95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

1994 (1)

1990 (1)

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

Aldred, M. P.

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

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]

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

Barozzi, S.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[CrossRef] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

Belov, V.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[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]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bossi, M.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

Bowman, G. R.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

Chang, E.

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

Chen, Z.

Dakin, K.

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[CrossRef] [PubMed]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[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]

Denk, W.

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 microm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003).
[CrossRef] [PubMed]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A.95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

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

Diaspro, A.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[CrossRef] [PubMed]

Drezek, R. A.

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

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.

Eggeling, C.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

Egner, A.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

Faretta, M.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[CrossRef] [PubMed]

Fernández-Suárez, M.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008).
[CrossRef] [PubMed]

Fölling, J.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

Fukami, K.

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]

Gee, K.

T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

Girirajan, T. P.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Goley, E. D.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

Hama, 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]

Hasan, M. T.

Heilemann, 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]

Hell, S. W.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett.19(11), 780–782 (1994).
[CrossRef] [PubMed]

Helmchen, F.

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

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A.95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Hofkens, J.

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]

Holfeld, B. 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]

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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).
[CrossRef] [PubMed]

Iwanaga, S.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[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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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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D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A.95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Kobat, D.

Kunetsky, R.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[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]

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H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
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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).
[CrossRef] [PubMed]

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M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

Li, C.

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

Li, W.-H.

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[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).
[CrossRef] [PubMed]

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E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

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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]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

Liu, F.

Lord, S. J.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

Mack-Bucher, J. A.

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]

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T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

Martinez, M. L.

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[CrossRef] [PubMed]

Mason, M. D.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

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J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[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]

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]

Min, W.

Mitchison, T. J.

T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

Mitra, P. P.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A.95(26), 15741–15746 (1998).
[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).
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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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H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[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]

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E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[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]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

Rao, J.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[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]

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M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[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]

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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]

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T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

Schaffer, C. B.

Schneider, M.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[CrossRef] [PubMed]

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J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

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H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[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]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

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

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M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[CrossRef] [PubMed]

Theer, P.

Theriot, J. A.

T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

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M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008).
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H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

Wang, H.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

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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]

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]

Wichmann, J.

Williams, J. C.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[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]

Wong, A. W.

Xie, H.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

Xu, C.

Xu, K.

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[CrossRef] [PubMed]

Ying, J.

Zhan, K.

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

Zhang, G.-F.

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

Zhao, Y.

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[CrossRef] [PubMed]

Zheng, Q.

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[CrossRef] [PubMed]

Zhu, M.-Q.

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[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]

Angew. Chem. Int. Ed. Engl. (1)

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, “Photochromic rhodamines provide nanoscopy with optical sectioning,” Angew. Chem. Int. Ed. Engl.46(33), 6266–6270 (2007).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biomed. Opt. Express (1)

Biophys. J. (3)

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, “Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,” Biophys. J.89(2), 1346–1352 (2005).
[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]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (3)

Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, and W.-H. Li, “New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications,” J. Am. Chem. Soc.126(14), 4653–4663 (2004).
[CrossRef] [PubMed]

H. L. Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H. Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J. Rao, and W. E. Moerner, “Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores,” J. Am. Chem. Soc.132(43), 15099–15101 (2010).
[CrossRef] [PubMed]

M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek, and A. D. Q. Li, “Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles Targeted to Cancer Cells,” J. Am. Chem. Soc.133(2), 365–372 (2011).
[CrossRef]

J. Biomed. Opt. (2)

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 (1)

Laser Photon. Rev. (1)

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]

Methods Enzymol. (1)

T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee, and A. Mallavarapu, “Caged fluorescent probes,” Methods Enzymol.291, 63–78 (1998).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

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 (3)

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

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[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).
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Nat. Neurosci. (1)

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Figures (7)

Fig. 1
Fig. 1

Fundamental imaging-depth limit of multi-photon fluorescence microscopy. (a) Images of a tissue phantom consisting of 5% intralipid, 1 µm fluorescent beads (Invitrogen F8765) and 1% agarose gel under a constant excitation laser power. Two-photon fluorescence signal quickly attenuates with the imaging depth. (b) Images of the same sample using a compensative higher laser power to maintain the signal strength at different depths. The resulting images, although showing signals deeper into the sample, suffer from a loss of contrast as the out-of-focus background grows. 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

Imaging contrast of multi-photon microscopy depends on both sample scattering and background fluorophore concentration. A set of “two-layer” samples (fluorescent beads were placed on a glass coverslip as the target, while a thick layer of mixed fluorescein dye solution and scattering intralipids was inserted as the background between the target and the objective.) with varying intralipid contents and background dye concentrations were imaged using a two-photon microscope. The laser power was set higher accordingly when a more scattering sample was imaged. At a given background layer thickness, image contrast deteriorates only when both a significant background turbidity and a dense background fluorophore staining are present. Imaging contrast further deteriorates when thicker background layers were applied.

Fig. 3
Fig. 3

Principle of multiphoton activation and imaging (MPAI). (a) When imaging transparent samples, fluorescence is only generated at the laser focus where the intensity is the highest. (b) When imaging deep into scattering samples, substantial laser intensities are distributed out of focus, generating background fluorescence that is comparable to or even stronger than the in-focus signal. (c) When imaging with PAFs which are originally in the dark state, the multiphoton activation will switch on a higher percentage of PAFs at focus than those out-of-focus. Such a spatial disparity of dark-bright transitions would lead to a significantly decreased background fluorescence in the subsequent imaging.

Fig. 4
Fig. 4

Experimental demonstration of MPAI with caged fluorescein. (a) CMNB (5-carboxymethoxy-2-nitrobenzyl) caging groups of CMNB-caged fluorescein could be cleaved by a 750 nm pulsed laser, leading to the formation of fluorescein in the bright state, which could be excited by the same laser to emit fluorescence. (b) Absorption and fluorescence spectra of caged fluorescein and uncaged fluorescein, with the activation kinetics under UV illumination. A ~100-fold fluorescence enhancement was observed after a complete activation. (c-f) Imaging “two-layer” samples (Fig. 7.) where the targets are 2 µL droplets of 1 mM dye solution and the background layers (120 µm thick) consist of scattering polystyrene beads (0.9 µm) and dye solution (1 mM for (c) and (d), 3 mM for (e) and (f)). Boundaries of liquid droplets cross the field of view so that the darker parts correspond to the background while the brighter parts represent the sum of the signal and the background. When the imaging depth-limit is reached for regular fluorescein in (c) with a S/B of about 1.2, a 20-times improvement is achieved for caged fluorescein in (d). With a three folds more dyes in the background layer of (e) and (f), imaging contrast becomes extremely poor for regular fluorescein, while the caged fluorescein still offers a S/B of about 8.

Fig. 5
Fig. 5

Experimental demonstration of MPAI with pa-GFP. (a) pa-GFP could be activated by a pulsed laser at 830 nm to its bright state, which could be further excited by a 920 nm pulsed laser to emit fluorescence. (b, c) Deep imaging comparison of 3D turbid samples made of E. coli cells expressing free regular GFP (b) or pa-GFP (c) embedded in 2% agarose gel with the same cell densities. While out-of-focus background is overwhelming when imaging E. coli expressing regular GFP at a 100 µm depth inside the gel, MPAI with pa-GFP at the same depth offers a satisfactory image contrast.

Fig. 6
Fig. 6

The “two-layer” sample design for beads imaging experiments. Fluorescent beads were placed on a glass coverslip as the target, while a thick layer of mixed fluorescein dye solution with scattering intralipid was inserted as the background between the target and the objective. From left to right, the background fluorescein concentration increases; from top to bottom, the scattering intralipid percentage increases. The signal-to-background ratio decreases with the increase of both the background scattering and the background dye concentration.

Fig. 7
Fig. 7

The “two-layer” sample design for droplet imaging experiments. (a): imaging with caged fluorescein (Fig. 4f). (b): imaging with regular fluorescein (Fig. 4e). Images were taken across the boundaries of caged-fluorescein or regular fluorescein droplets on top of a layer of caged-fluorescein or regular fluorescein solution doped with scattering polystyrene beads.

Equations (9)

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( S B ) regular = V in 0 τ C S (r,z) I i 2 (r,z,t)dtdV V out 0 τ C B (r,z) I i 2 (r,z,t)dtdV =1,
A(r,z,t)=C(r,z)η(r,z,t).
η(t)=1exp[ σ Ι a 2 (r,z,t)t ] 0tτ,
( S B ) PAFs = V in 0 τ C S (r,z) I i 2 [ 1 e σ I a 2 (r,z,t)t ] I i 2 (r,z,t)dtdV V out 0 τ C B (r,z)[ 1 e σ I a 2 (r,z,t)t ] I i 2 (r,z,t)dtdV .
(S/B) PAFs > (S/B) regular.
( N S N B ) activated = V in 0 τ C S (r,z) [ 1 e σ I a 2 (r,z,t)τ ]dtdV V out 0 τ C B (r,z)[ 1 e σ I a 2 (r,z,t)τ ]dtdV . V in 0 τ C S (r,z) I a 2 (r,z,t)dtdV V out 0 τ C B (r,z) I a 2 (r,z,t)dtdV =1.
( N S N B ) regular = V in C S (r,z)dV V out C B (r,z)dV V in V out 1.
( S B ) PAFs = V in 0 τ C S (r,z) η S (r,z,t) I i 2 (r,z,t)dtdV V out 0 τ C B (r,z) η B (r,z,f(z)·τ) I i 2 (r,z,t)dtdV .
η S (r,z,τ) η B (r,z,f(z)·τ) I a 2 (r,z) | V in f(z)· I a 2 (r,z) | V out in I i 2 (r,z)dr out I a 2 (r,z)dr >1.

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