Abstract

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep MPM has never before been demonstrated. Herein, we characterize the multiphoton properties of three pdot variants and perform deep in vivo MPM imaging of cortical rodent microvasculature. We find pdot brightness exceeds conventional fluorophores, including quantum dots, and their broad multiphoton absorption spectrum permits imaging at wavelengths better-suited for biological imaging and confers compatibility with a range of longer excitation wavelengths. This results in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths (z = 1,300 µm). Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, enabling interrogation of deep structures in vivo.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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2018 (1)

2017 (5)

D. R. Miller, J. W. Jarrett, A. M. Hassan, and A. K. Dunn, “Deep tissue imaging with multiphoton fluorescence microscopy,” Curr Opin Biomed Eng 4, 32–39 (2017).
[Crossref] [PubMed]

D. R. Miller, A. M. Hassan, J. W. Jarrett, F. A. Medina, E. P. Perillo, K. Hagan, S. M. Shams Kazmi, T. A. Clark, C. T. Sullender, T. A. Jones, B. V. Zemelman, and A. K. Dunn, “In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure,” Biomed. Opt. Express 8(7), 3470–3481 (2017).
[Crossref] [PubMed]

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

J. Yu, Y. Rong, C.-T. Kuo, X.-H. Zhou, and D. T. Chiu, “Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine,” Anal. Chem. 89(1), 42–56 (2017).
[Crossref] [PubMed]

2016 (1)

2015 (2)

M. Massey, M. Wu, E. M. Conroy, and W. R. Algar, “Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications,” Curr. Opin. Biotechnol. 34, 30–40 (2015).
[Crossref] [PubMed]

C. J. Schrandt, S. M. Kazmi, T. A. Jones, and A. K. Dunn, “Chronic Monitoring of Vascular Progression after Ischemic Stroke Using Multiexposure Speckle Imaging and Two-Photon Fluorescence Microscopy,” J. Cereb. Blood Flow Metab. 35(6), 933–942 (2015).
[Crossref] [PubMed]

2014 (1)

2013 (6)

S. M. S. Kazmi, A. J. Salvaggio, A. D. Estrada, M. A. Hemati, N. K. Shaydyuk, E. Roussakis, T. A. Jones, S. A. Vinogradov, and A. K. Dunn, “Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion,” Biomed. Opt. Express 4(7), 1061–1073 (2013).
[Crossref] [PubMed]

C. Xu and F. W. Wise, “Recent advances in fibre lasers for nonlinear microscopy,” Nat. Photonics 7(11), 875–882 (2013).
[Crossref] [PubMed]

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

S. L. Jacques, “Optical properties of biological tissues: a review,” Phys. Med. Biol. 58(11), R37–R61 (2013).
[Crossref] [PubMed]

C. Wu and D. T. Chiu, “Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine,” Angew. Chem. Int. Ed. Engl. 52(11), 3086–3109 (2013).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

2011 (2)

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

Z. Hashim, P. Howes, and M. Green, “Luminescent quantum-dot-sized conjugated polymernanoparticles—nanoparticle formation in a miniemulsion system,” J. Mater. Chem. 21(6), 1797–1803 (2011).
[Crossref]

2010 (2)

J. Pecher and S. Mecking, “Nanoparticles of conjugated polymers,” Chem. Rev. 110(10), 6260–6279 (2010).
[Crossref] [PubMed]

A. Kaeser and A. P. H. J. Schenning, “Fluorescent Nanoparticles Based on Self-Assembled π-Conjugated Systems,” Adv. Mater. 22(28), 2985–2997 (2010).
[Crossref] [PubMed]

2008 (3)

A. D. Estrada, A. Ponticorvo, T. N. Ford, and A. K. Dunn, “Microvascular oxygen quantification using two-photon microscopy,” Opt. Lett. 33(10), 1038–1040 (2008).
[Crossref] [PubMed]

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]

U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008).
[Crossref] [PubMed]

2007 (1)

C. Wu, C. Szymanski, Z. Cain, and J. McNeill, “Conjugated Polymer Dots for Multiphoton Fluorescence Imaging,” J. Am. Chem. Soc. 129(43), 12904–12905 (2007).
[Crossref] [PubMed]

2006 (1)

R. Hardman, “A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors,” Environ. Health Perspect. 114(2), 165–172 (2006).
[Crossref] [PubMed]

2005 (1)

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

2004 (2)

A. M. Derfus, W. C. W. Chan, and S. N. Bhatia, “Probing the Cytotoxicity Of Semiconductor Quantum Dots,” Nano Lett. 4(1), 11–18 (2004).
[Crossref] [PubMed]

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
[Crossref] [PubMed]

2003 (2)

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

J. V. Frangioni, “In vivo near-infrared fluorescence imaging,” Curr. Opin. Chem. Biol. 7(5), 626–634 (2003).
[Crossref] [PubMed]

2001 (1)

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

1998 (3)

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]

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

M. A. Albota, C. Xu, and W. W. Webb, “Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm,” Appl. Opt. 37(31), 7352–7356 (1998).
[Crossref] [PubMed]

1996 (1)

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. 13(3), 481–491 (1996).
[Crossref]

1994 (1)

S. A. Soper and Q. L. Mattingly, “Steady-State and Picosecond Laser Fluorescence Studies of Nonradiative Pathways in Tricarbocyanine Dyes: Implications to the Design of Near-IR Fluorochromes with High Fluorescence Efficiencies,” J. Am. Chem. Soc. 116(9), 3144–3152 (1994).
[Crossref]

1993 (1)

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, and E. B. Shera, “The Photophysical Constants of Several Fluorescent Dyes Pertaining to Ultrasensitive Fluorescence Spectroscopy,” Photochem. Photobiol. 57(s1), 972–977 (1993).
[Crossref]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Albota, M.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Albota, M. A.

Algar, W. R.

M. Massey, M. Wu, E. M. Conroy, and W. R. Algar, “Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications,” Curr. Opin. Biotechnol. 34, 30–40 (2015).
[Crossref] [PubMed]

Beljonne, D.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Bhatia, S. N.

A. M. Derfus, W. C. W. Chan, and S. N. Bhatia, “Probing the Cytotoxicity Of Semiconductor Quantum Dots,” Nano Lett. 4(1), 11–18 (2004).
[Crossref] [PubMed]

Bonteanu, A.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

Brédas, J.-L.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Brown, E. B.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Bruchez, M. P.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

Burnham, D. R.

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

Cain, Z.

C. Wu, C. Szymanski, Z. Cain, and J. McNeill, “Conjugated Polymer Dots for Multiphoton Fluorescence Imaging,” J. Am. Chem. Soc. 129(43), 12904–12905 (2007).
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Campbell, R. B.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Carmeliet, P.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Cavaliere-Jaricot, S.

U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008).
[Crossref] [PubMed]

Chan, W. C. W.

A. M. Derfus, W. C. W. Chan, and S. N. Bhatia, “Probing the Cytotoxicity Of Semiconductor Quantum Dots,” Nano Lett. 4(1), 11–18 (2004).
[Crossref] [PubMed]

Chen, S.-J.

Cheng, L.-C.

Cheng, Y.-T.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Chiu, D. T.

J. Yu, Y. Rong, C.-T. Kuo, X.-H. Zhou, and D. T. Chiu, “Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine,” Anal. Chem. 89(1), 42–56 (2017).
[Crossref] [PubMed]

C. Wu and D. T. Chiu, “Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine,” Angew. Chem. Int. Ed. Engl. 52(11), 3086–3109 (2013).
[Crossref] [PubMed]

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

Clark, C. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Clark, S. W.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

Clark, T. A.

Conroy, E. M.

M. Massey, M. Wu, E. M. Conroy, and W. R. Algar, “Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications,” Curr. Opin. Biotechnol. 34, 30–40 (2015).
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Cruz-Hernández, J. C.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Davis, L. M.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, and E. B. Shera, “The Photophysical Constants of Several Fluorescent Dyes Pertaining to Ultrasensitive Fluorescence Spectroscopy,” Photochem. Photobiol. 57(s1), 972–977 (1993).
[Crossref]

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]

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,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Derfus, A. M.

A. M. Derfus, W. C. W. Chan, and S. N. Bhatia, “Probing the Cytotoxicity Of Semiconductor Quantum Dots,” Nano Lett. 4(1), 11–18 (2004).
[Crossref] [PubMed]

Dunn, A. K.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

D. R. Miller, J. W. Jarrett, A. M. Hassan, and A. K. Dunn, “Deep tissue imaging with multiphoton fluorescence microscopy,” Curr Opin Biomed Eng 4, 32–39 (2017).
[Crossref] [PubMed]

D. R. Miller, A. M. Hassan, J. W. Jarrett, F. A. Medina, E. P. Perillo, K. Hagan, S. M. Shams Kazmi, T. A. Clark, C. T. Sullender, T. A. Jones, B. V. Zemelman, and A. K. Dunn, “In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure,” Biomed. Opt. Express 8(7), 3470–3481 (2017).
[Crossref] [PubMed]

E. P. Perillo, J. E. McCracken, D. C. Fernée, J. R. Goldak, F. A. Medina, D. R. Miller, H.-C. Yeh, and A. K. Dunn, “Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser,” Biomed. Opt. Express 7(2), 324–334 (2016).
[Crossref] [PubMed]

C. J. Schrandt, S. M. Kazmi, T. A. Jones, and A. K. Dunn, “Chronic Monitoring of Vascular Progression after Ischemic Stroke Using Multiexposure Speckle Imaging and Two-Photon Fluorescence Microscopy,” J. Cereb. Blood Flow Metab. 35(6), 933–942 (2015).
[Crossref] [PubMed]

S. M. S. Kazmi, A. J. Salvaggio, A. D. Estrada, M. A. Hemati, N. K. Shaydyuk, E. Roussakis, T. A. Jones, S. A. Vinogradov, and A. K. Dunn, “Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion,” Biomed. Opt. Express 4(7), 1061–1073 (2013).
[Crossref] [PubMed]

A. D. Estrada, A. Ponticorvo, T. N. Ford, and A. K. Dunn, “Microvascular oxygen quantification using two-photon microscopy,” Opt. Lett. 33(10), 1038–1040 (2008).
[Crossref] [PubMed]

Ehrlich, J. E.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Estrada, A. D.

Feng, D. D.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Feng, Y.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
[Crossref] [PubMed]

Fernée, D. C.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

E. P. Perillo, J. E. McCracken, D. C. Fernée, J. R. Goldak, F. A. Medina, D. R. Miller, H.-C. Yeh, and A. K. Dunn, “Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser,” Biomed. Opt. Express 7(2), 324–334 (2016).
[Crossref] [PubMed]

Fertig, N.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

Ford, T. N.

Frangioni, J. V.

J. V. Frangioni, “In vivo near-infrared fluorescence imaging,” Curr. Opin. Chem. Biol. 7(5), 626–634 (2003).
[Crossref] [PubMed]

Fu, J.-Y.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Fukumura, D.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Gallina, M. E.

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

Gaub, H. E.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

Goldak, J. R.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

E. P. Perillo, J. E. McCracken, D. C. Fernée, J. R. Goldak, F. A. Medina, D. R. Miller, H.-C. Yeh, and A. K. Dunn, “Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser,” Biomed. Opt. Express 7(2), 324–334 (2016).
[Crossref] [PubMed]

Grabolle, M.

U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008).
[Crossref] [PubMed]

Green, M.

Z. Hashim, P. Howes, and M. Green, “Luminescent quantum-dot-sized conjugated polymernanoparticles—nanoparticle formation in a miniemulsion system,” J. Mater. Chem. 21(6), 1797–1803 (2011).
[Crossref]

Hagan, K.

Hansen, S. J.

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

Hardman, R.

R. Hardman, “A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors,” Environ. Health Perspect. 114(2), 165–172 (2006).
[Crossref] [PubMed]

Hashim, Z.

Z. Hashim, P. Howes, and M. Green, “Luminescent quantum-dot-sized conjugated polymernanoparticles—nanoparticle formation in a miniemulsion system,” J. Mater. Chem. 21(6), 1797–1803 (2011).
[Crossref]

Hassan, A.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

Hassan, A. M.

Heikal, A. A.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Helmchen, F.

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]

Hemati, M. A.

Hess, S. E.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Horton, N. G.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

L.-C. Cheng, N. G. Horton, K. Wang, S.-J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Hou, Q.

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

Howes, P.

Z. Hashim, P. Howes, and M. Green, “Luminescent quantum-dot-sized conjugated polymernanoparticles—nanoparticle formation in a miniemulsion system,” J. Mater. Chem. 21(6), 1797–1803 (2011).
[Crossref]

Hu, J. Y.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
[Crossref] [PubMed]

Jacques, S. L.

S. L. Jacques, “Optical properties of biological tissues: a review,” Phys. Med. Biol. 58(11), R37–R61 (2013).
[Crossref] [PubMed]

Jain, R. K.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Jarrett, J. W.

D. R. Miller, J. W. Jarrett, A. M. Hassan, and A. K. Dunn, “Deep tissue imaging with multiphoton fluorescence microscopy,” Curr Opin Biomed Eng 4, 32–39 (2017).
[Crossref] [PubMed]

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

D. R. Miller, A. M. Hassan, J. W. Jarrett, F. A. Medina, E. P. Perillo, K. Hagan, S. M. Shams Kazmi, T. A. Clark, C. T. Sullender, T. A. Jones, B. V. Zemelman, and A. K. Dunn, “In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure,” Biomed. Opt. Express 8(7), 3470–3481 (2017).
[Crossref] [PubMed]

Jin, Y.

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

Jones, T. A.

Kaeser, A.

A. Kaeser and A. P. H. J. Schenning, “Fluorescent Nanoparticles Based on Self-Assembled π-Conjugated Systems,” Adv. Mater. 22(28), 2985–2997 (2010).
[Crossref] [PubMed]

Kazmi, S. M.

C. J. Schrandt, S. M. Kazmi, T. A. Jones, and A. K. Dunn, “Chronic Monitoring of Vascular Progression after Ischemic Stroke Using Multiexposure Speckle Imaging and Two-Photon Fluorescence Microscopy,” J. Cereb. Blood Flow Metab. 35(6), 933–942 (2015).
[Crossref] [PubMed]

Kazmi, S. M. S.

Keller, R. A.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, and E. B. Shera, “The Photophysical Constants of Several Fluorescent Dyes Pertaining to Ultrasensitive Fluorescence Spectroscopy,” Photochem. Photobiol. 57(s1), 972–977 (1993).
[Crossref]

Kerr, J. N. D.

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]

Kirchner, C.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

Kleinfeld, D.

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.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Kogej, T.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Kudera, S.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

Kuo, C.-T.

J. Yu, Y. Rong, C.-T. Kuo, X.-H. Zhou, and D. T. Chiu, “Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine,” Anal. Chem. 89(1), 42–56 (2017).
[Crossref] [PubMed]

Larson, D. R.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

Lee, L. Y.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
[Crossref] [PubMed]

Levin, M. D.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Li, B.

Liedl, T.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

Liu, Y.-L.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

Marder, S. R.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
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M. Massey, M. Wu, E. M. Conroy, and W. R. Algar, “Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications,” Curr. Opin. Biotechnol. 34, 30–40 (2015).
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S. A. Soper and Q. L. Mattingly, “Steady-State and Picosecond Laser Fluorescence Studies of Nonradiative Pathways in Tricarbocyanine Dyes: Implications to the Design of Near-IR Fluorochromes with High Fluorescence Efficiencies,” J. Am. Chem. Soc. 116(9), 3144–3152 (1994).
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M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
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McCracken, J. E.

McNeill, J.

C. Wu, C. Szymanski, Z. Cain, and J. McNeill, “Conjugated Polymer Dots for Multiphoton Fluorescence Imaging,” J. Am. Chem. Soc. 129(43), 12904–12905 (2007).
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C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
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Miller, D. R.

Mitra, P. P.

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C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
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Nann, T.

U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008).
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Ng, W. J.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
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Nishimura, N.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
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U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008).
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S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, and E. B. Shera, “The Photophysical Constants of Several Fluorescent Dyes Pertaining to Ultrasensitive Fluorescence Spectroscopy,” Photochem. Photobiol. 57(s1), 972–977 (1993).
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C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
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Ong, S. L.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
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D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
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C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

Pecher, J.

J. Pecher and S. Mecking, “Nanoparticles of conjugated polymers,” Chem. Rev. 110(10), 6260–6279 (2010).
[Crossref] [PubMed]

Pellegrino, T.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
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Perillo, E. P.

Perry, J. W.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
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Ponticorvo, A.

Reimer, J.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Resch-Genger, U.

U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008).
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Röckel, H.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
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Rong, Y.

J. Yu, Y. Rong, C.-T. Kuo, X.-H. Zhou, and D. T. Chiu, “Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine,” Anal. Chem. 89(1), 42–56 (2017).
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Roussakis, E.

Rumi, M.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
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Salvaggio, A. J.

Schaffer, C. B.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
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Shaydyuk, N. K.

Shera, E. B.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, and E. B. Shera, “The Photophysical Constants of Several Fluorescent Dyes Pertaining to Ultrasensitive Fluorescence Spectroscopy,” Photochem. Photobiol. 57(s1), 972–977 (1993).
[Crossref]

Sinefeld, D.

Soper, S. A.

S. A. Soper and Q. L. Mattingly, “Steady-State and Picosecond Laser Fluorescence Studies of Nonradiative Pathways in Tricarbocyanine Dyes: Implications to the Design of Near-IR Fluorochromes with High Fluorescence Efficiencies,” J. Am. Chem. Soc. 116(9), 3144–3152 (1994).
[Crossref]

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, and E. B. Shera, “The Photophysical Constants of Several Fluorescent Dyes Pertaining to Ultrasensitive Fluorescence Spectroscopy,” Photochem. Photobiol. 57(s1), 972–977 (1993).
[Crossref]

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E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

Stölzle, S.

C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H. E. Gaub, S. Stölzle, N. Fertig, and W. J. Parak, “Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles,” Nano Lett. 5(2), 331–338 (2005).
[Crossref] [PubMed]

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W. Denk, J. H. Strickler, and W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Subramaniam, G.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Sullender, C. T.

Sun, W.

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

Szymanski, C.

C. Wu, C. Szymanski, Z. Cain, and J. McNeill, “Conjugated Polymer Dots for Multiphoton Fluorescence Imaging,” J. Am. Chem. Soc. 129(43), 12904–12905 (2007).
[Crossref] [PubMed]

Tan, X.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
[Crossref] [PubMed]

Tolias, A. S.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Tsuzuki, Y.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Vinogradov, S. A.

Wang, K.

L.-C. Cheng, N. G. Horton, K. Wang, S.-J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Wang, M.

M. Wang, C. Wu, D. Sinefeld, B. Li, F. Xia, and C. Xu, “Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain,” Biomed. Opt. Express 9(8), 3534–3543 (2018).
[Crossref] [PubMed]

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Wang, T.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

Webb, W. W.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

M. A. Albota, C. Xu, and W. W. Webb, “Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm,” Appl. Opt. 37(31), 7352–7356 (1998).
[Crossref] [PubMed]

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. 13(3), 481–491 (1996).
[Crossref]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Williams, R. M.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

Wise, F. W.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

C. Xu and F. W. Wise, “Recent advances in fibre lasers for nonlinear microscopy,” Nat. Photonics 7(11), 875–882 (2013).
[Crossref] [PubMed]

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science 300(5624), 1434–1436 (2003).
[Crossref] [PubMed]

Wong, S. W.

L. Y. Lee, S. L. Ong, J. Y. Hu, W. J. Ng, Y. Feng, X. Tan, and S. W. Wong, “Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum,” Appl. Environ. Microbiol. 70(10), 5732–5736 (2004).
[Crossref] [PubMed]

Wu, C.

M. Wang, C. Wu, D. Sinefeld, B. Li, F. Xia, and C. Xu, “Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain,” Biomed. Opt. Express 9(8), 3534–3543 (2018).
[Crossref] [PubMed]

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

C. Wu and D. T. Chiu, “Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine,” Angew. Chem. Int. Ed. Engl. 52(11), 3086–3109 (2013).
[Crossref] [PubMed]

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
[Crossref] [PubMed]

C. Wu, C. Szymanski, Z. Cain, and J. McNeill, “Conjugated Polymer Dots for Multiphoton Fluorescence Imaging,” J. Am. Chem. Soc. 129(43), 12904–12905 (2007).
[Crossref] [PubMed]

Wu, M.

M. Massey, M. Wu, E. M. Conroy, and W. R. Algar, “Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications,” Curr. Opin. Biotechnol. 34, 30–40 (2015).
[Crossref] [PubMed]

Wu, X.-L.

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

Xia, F.

Xu, C.

M. Wang, C. Wu, D. Sinefeld, B. Li, F. Xia, and C. Xu, “Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain,” Biomed. Opt. Express 9(8), 3534–3543 (2018).
[Crossref] [PubMed]

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017).
[Crossref] [PubMed]

L.-C. Cheng, N. G. Horton, K. Wang, S.-J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

C. Xu and F. W. Wise, “Recent advances in fibre lasers for nonlinear microscopy,” Nat. Photonics 7(11), 875–882 (2013).
[Crossref] [PubMed]

M. Albota, D. Beljonne, J.-L. Brédas, J. E. Ehrlich, J.-Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X.-L. Wu, and C. Xu, “Design of Organic Molecules with Large Two-Photon Absorption Cross Sections,” Science 281(5383), 1653–1656 (1998).
[Crossref] [PubMed]

M. A. Albota, C. Xu, and W. W. Webb, “Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm,” Appl. Opt. 37(31), 7352–7356 (1998).
[Crossref] [PubMed]

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. 13(3), 481–491 (1996).
[Crossref]

Xu, L.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001).
[Crossref] [PubMed]

Ye, F.

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

Yeh, H.-C.

E. P. Perillo, J. W. Jarrett, Y.-L. Liu, A. Hassan, D. C. Fernée, J. R. Goldak, A. Bonteanu, D. J. Spence, H.-C. Yeh, and A. K. Dunn, “Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser,” Light Sci. Appl. 6(11), e17095 (2017).
[Crossref] [PubMed]

E. P. Perillo, J. E. McCracken, D. C. Fernée, J. R. Goldak, F. A. Medina, D. R. Miller, H.-C. Yeh, and A. K. Dunn, “Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser,” Biomed. Opt. Express 7(2), 324–334 (2016).
[Crossref] [PubMed]

Yu, J.

J. Yu, Y. Rong, C.-T. Kuo, X.-H. Zhou, and D. T. Chiu, “Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine,” Anal. Chem. 89(1), 42–56 (2017).
[Crossref] [PubMed]

W. Sun, F. Ye, M. E. Gallina, J. Yu, C. Wu, and D. T. Chiu, “Lyophilization of Semiconducting Polymer Dot Bioconjugates,” Anal. Chem. 85(9), 4316–4320 (2013).
[Crossref] [PubMed]

C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. D. McNeill, J. M. Olson, and D. T. Chiu, “Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting,” Angew. Chem. Int. Ed. Engl. 50(15), 3430–3434 (2011).
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Figures (10)

Fig. 1
Fig. 1 Polymer dot power dependence. (A) Logarithmic plots of the dependence of two- and three-photon induced fluorescence on excitation power. The excitation wavelength and fitted slope is indicated in the legend of each graph. The estimated uncertainty of each slope is reported as a standard deviation. Each plot corresponds to a distinct polymer dot species. (B) CNPPV (left), PFBT (middle), and PFPV (right) power dependence versus wavelength. The dashed and dash-dotted lines correspond to pure two- and three-photon power dependence, respectively. The blue shaded region represents the titanium-sapphire tuning range (λex = 700 – 1,000 nm), the yellow shaded region represents the ytterbium-fiber laser’s bandwidth (λex = 1,060 nm; Δλ = 40 nm), and the green shaded region represents the optical parametric amplifier tuning range (λex = 1,100 – 1,400 nm).
Fig. 2
Fig. 2 Titanium-sapphire in vivo vascular imaging of C57 mice. Reto-orbital injection labeling with polymer dots (Columns 1 −3), dextran-conjugated fluorescein (Column 4), and semiconductor quantum dots (QD605; Column 5); λex = 800 nm; scale bars = 100 µm. Maximum intensity projections spanning 100 µm ranges were taken at various depth intervals in the cortex, with distance relative to the pial surface indicated across the left margin. Polymer dots yield appreciably higher signal-to-background ratio at all depths relative to fluorescein and QD605. Imaging of QD605-labeled vasculature beyond 550 µm was restricted due to the toxicity of the injection. All depth comparisons stem from age-matched litter mates; CNPPV, PFBT, and fluorescein imaging was performed in the same specimen.
Fig. 3
Fig. 3 Two-photon imaging of PFPV polymer dots intravenously injected in C57 mice. (A) Laser speckle contrast image of surface blood vessels. (B) A tangential (xy) maximum intensity projection of a 365 × 365 × 850 µm3 image stack collected from the region of interest (ROI) delineated in red in panel A. (C) A max intensity projection of a 3D reconstruction from the same data. (D) 2D tangential (xy) projections over shorter depth ranges of the stack. Unlabeled scale bars = 100 µm.
Fig. 4
Fig. 4 Longer wavelength excitation (λex = 1,225 nm) versus shorter wavelength excitation (λex = 800 nm) of CNPPV-labeled C57 vasculature. (A) Sagittal (xz) projection of a 365 × 365 × 1,300 µm3 image stack at λex = 1,225 nm using an optical parametric amplifier source. (B) Sagittal (xz) projection of a 356 × 365 × 850 µm3 image stack of the same region show in A at λex = 800 nm using a titanium-sapphire source. (C) 2D tangential (xy) projections over shorter depth ranges of the stacks shown in A and B. (D) Comparison plots of signal-to-background ratio (SBR) versus depth (Left) and background intensity versus depth (Right). Scale bars = 100 µm.
Fig. 5
Fig. 5 High power fiber laser imaging of PFPV-labeled vasculature improves the SBR of images. (A) Image stacks of the same cortical region collected using a 1,060 nm ytterbium-fiber laser (left, 256 x 256 x 900 µm3) or a 800 nm Ti:S excitation source (right, 256 x 256 x 850 µm3). The depth at which maximum power output from the Ti:S laser was reached is marked by a black line (z = 600 µm). Scale bars = 75 µm. (B) 50 µm thick maximum intensity projections of the images stacks shown in A centered at 700 µm. The blue and red lines denote the positions of analyzed 45 µm long line profiles. Scale bars = 75 µm. (C) A plot of normalized signal intensity relative to position. (D) Left, a vessel line scan collected at a depth of 750 µm; scale bar = 75 µm. Right, the analyzed blood flow velocity is 1.29 ± 0.20 mm/sec; scale bar = 25 ms and 20 µm.
Fig. 6
Fig. 6 (A) Molecular structures of CNPPV (Left), PFBT (Center), and PFPV (Right). (B) Size distributions of CNPPV (21.89 nm), PFBT (31.47 nm), and PFPV (22.74 nm) as measured by dynamic light scattering. (C) Absorption (Left) and emission (Right) spectra of CNPPV, PFBT, and PFPV [17].
Fig. 7
Fig. 7 A 5W pump (Verdi, Coherent) is used to seed a Ti:Sapphire (Ti:S) oscillator (Mira 900, Coherent), which is then stretched by a modified external stretcher/compressor and amplified by a Ti:S regenerative amplifier (RegA, Coherent) seeded by a 18W pump (Verdi G18, Coherent). The amplified pulse is then converted to a longer wavelength by an optical parametric amplifier tunable over a 1,100 – 1,400 nm range. The OPA output is compressed by a set of prisms before entering the scanning optics resulting in an objective-focused beam with a pulse width of ~45 fs.
Fig. 8
Fig. 8 Normalized signal-to-background (SBR) ratio versus depth comparison of polymer dots relative to fluorescein and QD605; λex = 800 nm. SBR is diminished at the surface due to high background signal from the dura resulting from second harmonic generation of collagen. At ~150 µm SBR is at a maximum for all contrast agents and gradually decreases with depth. Relative to QD605 and fluorescein, all three polymer dots retain a higher SBR beyond ~150 µm. To ensure fair comparisons, the average laser power at each depth was maintained at consistent levels across the separate imaging experiments.
Fig. 9
Fig. 9 An ideal biological imaging wavelength is situated at 1,300 nm [22,35]. The blue line indicates the photon fraction at a 1 mm depth in brain tissue versus wavelength. The red line indicates the percent of photon absorbed versus wavelength. The wavelength excitation regions of the titanium-sapphire (beige), ytterbium-fiber laser (gray), and optical parametric amplifier (blue) laser systems are delineated by color.
Fig. 10
Fig. 10 Semilog plot of polymer dot brightness relative to quantum dots (QDs), organic dyes (ODs), and fluorescent proteins (FPs). The two-photon action cross-sections of polymer dots are plotted as circles connected by a solid line (PFPV in green, and MEH-PPV in red-orange) [20]. The blue shaded region corresponds to the expected range of quantum dots’ two-photon action (2PA) cross sections [10,30]. The wavelength-dependent 2PA cross section of an atypically bright quantum dot species, QD605, is represented by black triangles with a dashed and dotted line [30]. The red shaded region corresponds to the expected range of 2PA cross sections of fluorescent dyes and proteins [10]. The 2PA cross sections of two typical fluorescent dyes are represented by diamonds connected by dashed lines (rhodamine B in magenta, fluorescein in dark green) and the 2PA cross sections of two typical fluorescent proteins are represented by squares connected by dotted lines (eGFP in blue and YFP in black) [40].

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