Abstract

Deep-tissue penetration is highly required in in vivo optical bioimaging. We synthesized a type of red emissive fluorophore BT with aggregation-induced emission (AIE) property. BT molecules were then encapsulated with amphiphilic polymers to form nanodots, and a large two-photon absorption (2PA) cross-section of 2.9 × 106 GM at 1040 nm was observed from each BT nanodot, which was much larger than those at the wavelengths of 770 to 860 nm. In addition, 1040 nm light was found to have better penetration and focusing capability than 800 nm light in biological tissue, according to the Monte Carlo simulation. The toxicity and tissue distribution of BT nanodots were studied, and they were found to have good biocompatibility. BT nanodots were then utilized for in vivo imaging of mouse ear and brain, and an imaging depth of 700 μm was obtained with the femtosecond (fs) excitation of 1040 nm. The red emissive AIE nanodots with high 2PA efficiency at 1040 nm would be useful for deep-tissue functional bioimaging in the future.

© 2015 Optical Society of America

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  4. S. Ogawa, T. M. Lee, A. R. Kay, and D. W. Tank, “Brain magnetic resonance imaging with contrast dependent on blood oxygenation,” Proc. Natl. Acad. Sci. U.S.A. 87(24), 9868–9872 (1990).
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  5. I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
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  10. M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
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    [Crossref] [PubMed]
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    [Crossref]
  23. D. Wang, J. Qian, S. He, J. S. Park, K. S. Lee, S. Han, and Y. Mu, “Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging,” Biomaterials 32(25), 5880–5888 (2011).
    [Crossref] [PubMed]
  24. D. Wang, J. Qian, W. Qin, A. Qin, B. Z. Tang, and S. He, “Biocompatible and photostable AIE dots with red emission for in vivo two-photon bioimaging,” Sci. Rep. 4, 4279 (2014).
    [PubMed]
  25. F. Cai, J. Yu, and S. He, “Vectorial electric field Monte Caro simulations for focused laser beams (800 nm-2220 nm) in a biological sample,” Prog. Electromagnetics Res. 142, 667–681 (2013).
    [Crossref]
  26. C. F. A. Gomez-Duran, R. Hu, G. Feng, T. Li, F. Bu, M. Arseneault, B. Liu, E. Peña-Cabrera, and B. Z. Tang, “Effect of AIE substituents on the fluorescence of tetraphenylethene-containing BODIPY derivatives,” ACS Appl. Mater. Interfaces 7(28), 15168–15176 (2015).
    [Crossref] [PubMed]
  27. P. Liu, S. Li, Y. Jin, L. Qian, N. Gao, S. Q. Yao, F. Huang, Q. H. Xu, and Y. Cao, “Red-Emitting DPSB-Based Conjugated Polymer Nanoparticles with High Two-Photon Brightness for Cell Membrane Imaging,” ACS Appl. Mater. Interfaces 7(12), 6754–6763 (2015).
    [Crossref] [PubMed]
  28. J. Geng, C. C. Goh, N. Tomczak, J. Liu, R. Liu, L. Ma, L. G. Ng, G. G. Gurzadyan, and B. Liu, “Micelle/Silica Co-protected Conjugated Polymer Nanoparticles for Two-Photon Excited Brain Vascular Imaging,” Chem. Mater. 26(5), 1874–1880 (2014).
    [Crossref]
  29. D. A. Oulianov, I. V. Tomov, A. S. Dvornikov, and P. M. Rentzepis, “Observations on the measurement of two-photon absorption cross-section,” Opt. Commun. 191(3–6), 235–243 (2001).
    [Crossref]
  30. N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express 16(6), 4029–4047 (2008).
    [Crossref] [PubMed]
  31. 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]
  32. H. Ding, J. Q. Lu, W. A. Wooden, P. J. Kragel, and X. H. Hu, “Refractive indices of human skin tissues at eight wavelengths and estimated dispersion relations between 300 and 1600 nm,” Phys. Med. Biol. 51(6), 1479–1489 (2006).
    [Crossref] [PubMed]
  33. A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. Schwarzmaier, “Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range,” Phys. Med. Biol. 47(12), 2059–2073 (2002).
    [Crossref] [PubMed]
  34. R. Hu, C. F. A. Gómez-Durán, J. W. Y. Lam, J. L. Belmonte-Vázquez, C. Deng, S. Chen, R. Ye, E. Peña-Cabrera, Y. Zhong, K. S. Wong, and B. Z. Tang, “Synthesis, solvatochromism, aggregation-induced emission and cell imaging of tetraphenylethene-containing BODIPY derivatives with large Stokes shifts,” Chem. Commun. (Camb.) 48(81), 10099–10101 (2012).
    [Crossref] [PubMed]
  35. A. Loudet and K. Burgess, “BODIPY dyes and their derivatives: Syntheses and spectroscopic properties,” Chem. Rev. 107(11), 4891–4932 (2007).
    [Crossref] [PubMed]
  36. Y. Hong, J. W. Y. Lam, and B. Z. Tang, “Aggregation-induced emission: phenomenon, mechanism and applications,” Chem. Commun. (Camb.) 29, 4332–4353 (2009).
    [Crossref] [PubMed]
  37. R. Hu, E. Lager, A. Aguilar-Aguilar, J. Liu, J. W. Y. Lam, H. H. Y. Sung, I. D. Williams, Y. Zhong, K. S. Wong, E. Pena-Cabrera, and B. Z. Tang, “Twisted intramolecular charge transfer and aggregation-induced emission of BODIPY derivatives,” J. Phys. Chem. C 113(36), 15845–15853 (2009).
    [Crossref]
  38. D. Ding, C. C. Goh, G. Feng, Z. Zhao, J. Liu, R. Liu, N. Tomczak, J. Geng, B. Z. Tang, L. G. Ng, and B. Liu, “Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging,” Adv. Mater. 25(42), 6083–6088 (2013).
    [Crossref] [PubMed]
  39. Y. Yang, F. An, Z. Liu, X. Zhang, M. Zhou, W. Li, X. Hao, C. S. Lee, and X. Zhang, “Ultrabright and ultrastable near-infrared dye nanoparticles for in vitro and in vivo bioimaging,” Biomaterials 33(31), 7803–7809 (2012).
    [Crossref] [PubMed]
  40. W. Qin, D. Ding, J. Liu, W. Z. Yuan, Y. Hu, B. Liu, and B. Z. Tang, “Biocompatible Nanoparticles with Aggregation-Induced Emission Characteristics as Far-Red/Near-Infrared Fluorescent Bioprobes for In Vitro and In Vivo Imaging Applications,” Adv. Funct. Mater. 22(4), 771–779 (2012).
    [Crossref]
  41. A. T. R. Williams, S. A. Winfield, and J. N. Miller, “Relative fluorescence quantum yields using a computer controlled luminescence spectrometer,” Analyst (Lond.) 108(1290), 1067–1071 (1983).
    [Crossref]

2015 (2)

C. F. A. Gomez-Duran, R. Hu, G. Feng, T. Li, F. Bu, M. Arseneault, B. Liu, E. Peña-Cabrera, and B. Z. Tang, “Effect of AIE substituents on the fluorescence of tetraphenylethene-containing BODIPY derivatives,” ACS Appl. Mater. Interfaces 7(28), 15168–15176 (2015).
[Crossref] [PubMed]

P. Liu, S. Li, Y. Jin, L. Qian, N. Gao, S. Q. Yao, F. Huang, Q. H. Xu, and Y. Cao, “Red-Emitting DPSB-Based Conjugated Polymer Nanoparticles with High Two-Photon Brightness for Cell Membrane Imaging,” ACS Appl. Mater. Interfaces 7(12), 6754–6763 (2015).
[Crossref] [PubMed]

2014 (4)

J. Geng, C. C. Goh, N. Tomczak, J. Liu, R. Liu, L. Ma, L. G. Ng, G. G. Gurzadyan, and B. Liu, “Micelle/Silica Co-protected Conjugated Polymer Nanoparticles for Two-Photon Excited Brain Vascular Imaging,” Chem. Mater. 26(5), 1874–1880 (2014).
[Crossref]

K. Li and B. Liu, “Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging,” Chem. Soc. Rev. 43(18), 6570–6597 (2014).
[Crossref] [PubMed]

X. Zhang, X. Zhang, L. Tao, Z. Chi, J. Xu, and Y. Wei, “Aggregation induced emission-based fluorescent nanoparticles: fabrication methodologies and biomedical applications,” J. Mater. Chem. B Mater. Biol. Med. 2(28), 4398–4414 (2014).
[Crossref]

D. Wang, J. Qian, W. Qin, A. Qin, B. Z. Tang, and S. He, “Biocompatible and photostable AIE dots with red emission for in vivo two-photon bioimaging,” Sci. Rep. 4, 4279 (2014).
[PubMed]

2013 (4)

F. Cai, J. Yu, and S. He, “Vectorial electric field Monte Caro simulations for focused laser beams (800 nm-2220 nm) in a biological sample,” Prog. Electromagnetics Res. 142, 667–681 (2013).
[Crossref]

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]

Y. Yang, Q. Zhao, W. Feng, and F. Li, “Luminescent chemodosimeters for bioimaging,” Chem. Rev. 113(1), 192–270 (2013).
[Crossref] [PubMed]

D. Ding, C. C. Goh, G. Feng, Z. Zhao, J. Liu, R. Liu, N. Tomczak, J. Geng, B. Z. Tang, L. G. Ng, and B. Liu, “Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging,” Adv. Mater. 25(42), 6083–6088 (2013).
[Crossref] [PubMed]

2012 (3)

Y. Yang, F. An, Z. Liu, X. Zhang, M. Zhou, W. Li, X. Hao, C. S. Lee, and X. Zhang, “Ultrabright and ultrastable near-infrared dye nanoparticles for in vitro and in vivo bioimaging,” Biomaterials 33(31), 7803–7809 (2012).
[Crossref] [PubMed]

W. Qin, D. Ding, J. Liu, W. Z. Yuan, Y. Hu, B. Liu, and B. Z. Tang, “Biocompatible Nanoparticles with Aggregation-Induced Emission Characteristics as Far-Red/Near-Infrared Fluorescent Bioprobes for In Vitro and In Vivo Imaging Applications,” Adv. Funct. Mater. 22(4), 771–779 (2012).
[Crossref]

R. Hu, C. F. A. Gómez-Durán, J. W. Y. Lam, J. L. Belmonte-Vázquez, C. Deng, S. Chen, R. Ye, E. Peña-Cabrera, Y. Zhong, K. S. Wong, and B. Z. Tang, “Synthesis, solvatochromism, aggregation-induced emission and cell imaging of tetraphenylethene-containing BODIPY derivatives with large Stokes shifts,” Chem. Commun. (Camb.) 48(81), 10099–10101 (2012).
[Crossref] [PubMed]

2011 (4)

D. Wang, J. Qian, S. He, J. S. Park, K. S. Lee, S. Han, and Y. Mu, “Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging,” Biomaterials 32(25), 5880–5888 (2011).
[Crossref] [PubMed]

Y. Hong, J. W. Y. Lam, and B. Z. Tang, “Aggregation-induced emission,” Chem. Soc. Rev. 40(11), 5361–5388 (2011).
[Crossref] [PubMed]

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

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

2009 (2)

Y. Hong, J. W. Y. Lam, and B. Z. Tang, “Aggregation-induced emission: phenomenon, mechanism and applications,” Chem. Commun. (Camb.) 29, 4332–4353 (2009).
[Crossref] [PubMed]

R. Hu, E. Lager, A. Aguilar-Aguilar, J. Liu, J. W. Y. Lam, H. H. Y. Sung, I. D. Williams, Y. Zhong, K. S. Wong, E. Pena-Cabrera, and B. Z. Tang, “Twisted intramolecular charge transfer and aggregation-induced emission of BODIPY derivatives,” J. Phys. Chem. C 113(36), 15845–15853 (2009).
[Crossref]

2008 (3)

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]

R. Ullah and J. Dutta, “Photocatalytic degradation of organic dyes with manganese-doped ZnO nanoparticles,” J. Hazard. Mater. 156(1-3), 194–200 (2008).
[Crossref] [PubMed]

N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express 16(6), 4029–4047 (2008).
[Crossref] [PubMed]

2007 (3)

N. J. Durr, T. Larson, D. K. Smith, B. A. Korgel, K. Sokolov, and A. Ben-Yakar, “Two-photon luminescence imaging of cancer cells using molecularly targeted gold nanorods,” Nano Lett. 7(4), 941–945 (2007).
[Crossref] [PubMed]

D. J. Brenner and E. J. Hall, “Computed tomography - an increasing source of radiation exposure,” N. Engl. J. Med. 357(22), 2277–2284 (2007).
[Crossref] [PubMed]

A. Loudet and K. Burgess, “BODIPY dyes and their derivatives: Syntheses and spectroscopic properties,” Chem. Rev. 107(11), 4891–4932 (2007).
[Crossref] [PubMed]

2006 (2)

H. Ding, J. Q. Lu, W. A. Wooden, P. J. Kragel, and X. H. Hu, “Refractive indices of human skin tissues at eight wavelengths and estimated dispersion relations between 300 and 1600 nm,” Phys. Med. Biol. 51(6), 1479–1489 (2006).
[Crossref] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

2005 (3)

H. Wang, T. B. Huff, D. A. Zweifel, W. He, P. S. Low, A. Wei, and J. X. Cheng, “In vitro and in vivo two-photon luminescence imaging of single gold nanorods,” Proc. Natl. Acad. Sci. U.S.A. 102(44), 15752–15756 (2005).
[Crossref] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
[Crossref] [PubMed]

2003 (1)

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]

2002 (2)

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
[Crossref] [PubMed]

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. Schwarzmaier, “Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range,” Phys. Med. Biol. 47(12), 2059–2073 (2002).
[Crossref] [PubMed]

2001 (4)

J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X. Zhan, Y. Liu, D. Zhu, and B. Z. Tang, “Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole,” Chem. Commun. (Camb.) 1740(18), 1740–1741 (2001).
[Crossref] [PubMed]

D. A. Oulianov, I. V. Tomov, A. S. Dvornikov, and P. M. Rentzepis, “Observations on the measurement of two-photon absorption cross-section,” Opt. Commun. 191(3–6), 235–243 (2001).
[Crossref]

K. T. Shimizu, R. G. Neuhauser, C. A. Leatherdale, S. A. Empedocles, W. K. Woo, and M. G. Bawendi, “Blinking statistics in single semiconductor nanocrystal quantum dots,” Phys. Rev. B 63(20), 205316 (2001).
[Crossref]

S. Link and M. A. El-Sayed, “Spectroscopic determination of the melting energy of a gold nanorod,” J. Chem. Phys. 114(5), 2362–2368 (2001).
[Crossref]

1999 (1)

J. Ophir, S. K. Alam, B. Garra, F. Kallel, E. Konofagou, T. Krouskop, and T. Varghese, “Elastography: ultrasonic estimation and imaging of the elastic properties of tissues,” Proc. Inst. Mech. Eng. H 213(3H3), 203–233 (1999).
[Crossref] [PubMed]

1998 (1)

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]

1990 (1)

S. Ogawa, T. M. Lee, A. R. Kay, and D. W. Tank, “Brain magnetic resonance imaging with contrast dependent on blood oxygenation,” Proc. Natl. Acad. Sci. U.S.A. 87(24), 9868–9872 (1990).
[Crossref] [PubMed]

1983 (1)

A. T. R. Williams, S. A. Winfield, and J. N. Miller, “Relative fluorescence quantum yields using a computer controlled luminescence spectrometer,” Analyst (Lond.) 108(1290), 1067–1071 (1983).
[Crossref]

Aguilar-Aguilar, A.

R. Hu, E. Lager, A. Aguilar-Aguilar, J. Liu, J. W. Y. Lam, H. H. Y. Sung, I. D. Williams, Y. Zhong, K. S. Wong, E. Pena-Cabrera, and B. Z. Tang, “Twisted intramolecular charge transfer and aggregation-induced emission of BODIPY derivatives,” J. Phys. Chem. C 113(36), 15845–15853 (2009).
[Crossref]

Alam, S. K.

J. Ophir, S. K. Alam, B. Garra, F. Kallel, E. Konofagou, T. Krouskop, and T. Varghese, “Elastography: ultrasonic estimation and imaging of the elastic properties of tissues,” Proc. Inst. Mech. Eng. H 213(3H3), 203–233 (1999).
[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]

An, F.

Y. Yang, F. An, Z. Liu, X. Zhang, M. Zhou, W. Li, X. Hao, C. S. Lee, and X. Zhang, “Ultrabright and ultrastable near-infrared dye nanoparticles for in vitro and in vivo bioimaging,” Biomaterials 33(31), 7803–7809 (2012).
[Crossref] [PubMed]

Arseneault, M.

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Figures (7)

Fig. 1
Fig. 1 (a) A scheme illustrating the synthesis process of BT nanodots. (b) Schematic illustration of the setup for 2PA cross-section measurements.
Fig. 2
Fig. 2 (a) Chemical structure and molecular geometry of BT. (b) and (c) Fluorescence spectra of BT in THF/water mixtures with different volume fractions of water (fw), (b) fw = 0 to 60 vol%, (c) fw = 70 to 95 vol%. (d) and (e) Plot of peak fluorescence intensity of BT versus fw in THF/water mixtures, (d) fw = 0 to 60 vol%, (e) fw = 70 to 95 vol%. [BT] = 10 μM; excitation wavelength λex = 420 nm.
Fig. 3
Fig. 3 (a) A representative TEM image of BT nanodots. Scale bar: 200 nm . (b) Extinction and 1PL spectra of BT molecules in THF, λex = 420nm. (c) Extinction and 1PL spectra of BT nanodots in aqueous dispersion, λex = 420nm. (d) 2PA cross-section of BT nanodots at various wavelengths.
Fig. 4
Fig. 4 Simulation of the light intensity distribution of 1040 nm and 800 nm laser beams in biological tissue at various vertical depths. (a-d) The simulated focal spots of 1040 nm laser beam at depths of 200 μm, 400 μm, 600 μm and 800 μm. (e-h) The simulated focal spots of 800 nm laser beam at depths of 200 μm, 400 μm, 600 μm and 800 μm. (i) The simulated light intensity of focal spots of 1040 nm and 800 nm laser beams at depths of 200 μm, 400 μm, 600 μm and 800 μm. (j) The focal spot intensity ratio of 1040 nm to 800 nm laser beam at depths of 200 μm, 400 μm, 600 μm and 800 μm.
Fig. 5
Fig. 5 Biodistribution and clearance of BT nanodots in mice. (a)-(d) Fluorescence images of different organs of the control group (a), as well as the mice 3 h (b), 12 h (c), and 72 h (d) post injection of BT nanodots. (e) Fluorescence images of liver of the control group, as well as the mice 3 h, 6h, 12 h, 24 h, and 72 h post injection of BT nanodots. (f) Fluorescence intensities of different organs of the control group, as well as the mice 3 h, 6h, 12 h, 24 h, and 72 h post injection of BT nanodots.
Fig. 6
Fig. 6 Intravital 2PL imaging of BT nanodots stained ear blood vessels in the mouse, at various vertical depths: (a) 0 μm, (b) 20 μm, (c) 40 μm, (d) 60 μm, (e) 80 μm, (f) 100 μm, (g) 120 μm. (h) 3D reconstructed 2PL images. Scale bar: 100 μm.
Fig. 7
Fig. 7 Intravital 2PL imaging of BT nanodots stained brain blood vessels in the mouse at various vertical depths: (a) 0 μm, (b) 50 μm, (c) 100 μm, (d) 150 μm, (e) 200 μm, (f) 250 μm, (g) 300 μm, (h) 350 μm, (i) 400 μm, (j) 450 μm, (k) 500 μm, (l) 550 μm, (m) 600 μm, (n) 650 μm, (o) 700 μm.(p, q) 3D reconstructed 2PL images with different visual angles. Scale bar: 100 μm.

Equations (1)

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δ 1 δ 0 = F 1 η 0 c 0 n 0 F 0 η 1 c 1 n 1

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