Skip to main content

Toxicity assessment and long-term three-photon fluorescence imaging of bright aggregation-induced emission nanodots in zebrafish


Aggregation-induced emission (AIE) luminogen displays bright fluorescence and has photobleaching resistance in its aggregation state. It is an ideal fluorescent contrast agent for bioimaging. Multiphoton microscopy is an important tool for bioimaging since it possesses the ability to penetrate deep into biological tissues. Herein, we used AIE luminogen together with multiphoton microscopy for long-term imaging of zebrafish. A typical AIE luminogen, 2,3-bis(4-(phenyl(4- (1,2,2-triphenylvinyl) phenyl)amino)phenyl) fumaronitrile (TPE-TPA-FN or TTF), was encapsulated with 1,2-distearoyl-sn-glycero-3-phosphoethanola-mine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) to form nanodots that exhibited bright three-photon fluorescence under 1,560 nm-femtosecond (fs) laser excitation. The TTF-nanodots were chemically stable in a wide range of pH values and showed no in vivo toxicity in zebrafish according to a series of biological tests. The TTF-nanodots were microinjected into zebrafish embryos, and the different growth stages of the labeled embryos were monitored with a three-photon fluorescence microscope. TTF-nanodots could be traced inside the zebrafish body for as long as 120 hours. In addition, the TTF-nanodots were utilized to target the blood vessel of zebrafish, and three-photon fluorescence angiogram was performed. More importantly, these nanodots were highly resistant to photobleaching under 1,560 nm-fs excitation, allowing long-term imaging of zebrafish.

This is a preview of subscription content, access via your institution.


  1. [1]

    Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 2006, 312, 217–224.

    Article  Google Scholar 

  2. [2]

    Domaille, D. W.; Que, E. L.; Chang, C. J. Synthetic fluorescent sensors for studying the cell biology of metals. Nat. Chem. Biol. 2008, 4, 168–175.

    Article  Google Scholar 

  3. [3]

    Hidalgo, M.; Urbano, M.; Ortiz, I.; Demyda-Peyras, S.; Murabito, M. R.; Gá lvez, M. J.; Dorado, J. DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy. Theriogenology 2015, 84, 399–406.

    Article  Google Scholar 

  4. [4]

    Tsachaki, M.; Birk, J.; Egert, A.; Odermatt, A. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions. Biochim. Biophys. Acta 2015, 1853, 1672–1682.

    Article  Google Scholar 

  5. [5]

    Chin, W. W. L.; Thong, P. S. P.; Bhuvaneswari, R.; Soo, K. C.; Heng, P. W. S.; Olivo, M. In-vivo optical detection of cancer using chlorin e6–polyvinylpyrrolidone induced fluorescence imaging and spectroscopy. BMC Med. Imaging 2009, 9, 1.

    Article  Google Scholar 

  6. [6]

    Ling, X. X.; Zhang, S. J.; Shao, P.; Li, W. X.; Yang, L.; Ding, Y.; Xu, C.; Stella, N.; Bai, M. F. A novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors CB2R over CB1R. Biomaterials 2015, 57, 169–178.

    Article  Google Scholar 

  7. [7]

    Chan, M. M.; Gray, B. D.; Pak, K. Y.; Fong, D. Non-invasive in vivo imaging of arthritis in a collagen-induced murine model with phosphatidylserine-binding near-infrared (NIR) dye. Arthritis Res. Ther. 2015, 17, 50.

    Article  Google Scholar 

  8. [8]

    Zhou, B. J.; Liu, W. M.; Zhang, H. Y.; Wu, J. S.; Liu, S.; Xu, H. T.; Wang, P. F. Imaging of nucleolar RNA in living cells using a highly photostable deep-red fluorescent probe. Biosens. Bioelectron. 2015, 68, 189–196.

    Article  Google Scholar 

  9. [9]

    Weissleder, R. A clearer vision for in vivo imaging. Nat. Biotechnol. 2001, 19, 316–317.

    Article  Google Scholar 

  10. [10]

    Sevick-Muraca, E. M.; Houston, J. P.; Gurfinkel, M. Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents. Curr. Opin. Chem. Biol. 2002, 6, 642–650.

    Article  Google Scholar 

  11. [11]

    So, P. T. C.; Dong, C. Y.; Masters, B. R.; Berland, K. M. Two-photon excitation fluorescence microscopy. Annu. Rev. Biomed. Eng. 2000, 2, 399–429.

    Article  Google Scholar 

  12. [12]

    Kobat, D.; Durst, M. E.; Nishimura, N.; Wong, A. W.; Schaffer, C. B.; Xu, C. Deep tissue multiphoton microscopy using longer wavelength excitation. Opt. Express 2009, 17, 13354–13364.

    Article  Google Scholar 

  13. [13]

    Kobat, D.; Horton, N. G.; Xu, C. In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J. Biomed. Opt. 2011, 16, 106014.

    Article  Google Scholar 

  14. [14]

    Kim, H. M.; Cho, B. R. Small-molecule two-photon probes for bioimaging applications. Chem. Rev. 2015, 115, 5014–5055.

    Article  Google Scholar 

  15. [15]

    Li, D. D.; Zhang, Q.; Wang, X. C.; Li, S. L.; Zhou, H. P.; Wu, J. Y.; Tian, Y. P. Self-assembly of a series of thiocyanate complexes with high two-photon absorbing active in near-IR range and bioimaging applications. Dyes Pigm. 2015, 120, 175–183.

    Article  Google Scholar 

  16. [16]

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

    Article  Google Scholar 

  17. [17]

    Daly, C. J.; McGrath, J. C. Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol. Ther. 2003, 100, 101–118.

    Article  Google Scholar 

  18. [18]

    Chen, F. Q.; Gerion, D. Fluorescent CdSe/ZnS nanocrystalpeptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Lett. 2004, 4, 1827–1832.

    Article  Google Scholar 

  19. [19]

    Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M. Use of quantum dots for live cell imaging. Nat. Methods 2004, 1, 73–78.

    Article  Google Scholar 

  20. [20]

    Shah, B. S.; Clark, P. A.; Moioli, E. K.; Stroscio, M. A.; Mao, J. J. Labeling of mesenchymal stem cells by bioconjugated quantum dots. Nano Lett. 2007, 7, 3071–3079.

    Article  Google Scholar 

  21. [21]

    Meng, Y. G.; Liang, J.; Wong, W. L.; Chisholm, V. Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene 2000, 242, 201–207.

    Article  Google Scholar 

  22. [22]

    Drobizhev, M.; Makarov, N. S.; Tillo, S. E.; Hughes, T. E.; Rebane, A. Two-photon absorption properties of fluorescent proteins. Nat. Methods 2011, 8, 393–399.

    Article  Google Scholar 

  23. [23]

    Kim, H. M.; Cho, B. R. Two-photon materials with large two-photon cross sections. Structure-property relationship. Chem. Commun. 2009, 153–164.

    Google Scholar 

  24. [24]

    Qian, J.; Wang, D.; Cai, F. L.; Zhan, Q. Q.; Wang, Y. L.; He, S. L. Photosensitizer encapsulated organically modified silica nanoparticles for direct two-photon photodynamic therapy and in vivo functional imaging. Biomaterials 2012, 33, 4851–4860.

    Article  Google Scholar 

  25. [25]

    Palma, A.; Alvarez, L. A.; Scholz, D.; Frimannsson, D. O.; Grossi, M.; Quinn, S. J.; O’Shea, D. F. Cellular uptake mediated off/on responsive near-infrared fluorescent nanoparticles. J. Am. Chem. Soc. 2011, 133, 19618–19621.

    Article  Google Scholar 

  26. [26]

    Birks, J. B. Photophysics of Aromatic Molecules; Wiley- Interscience: London, 1970.

    Google Scholar 

  27. [27]

    Wagh, A.; Qian, S. Y.; Law, B. Development of biocompatible polymeric nanoparticles for in vivo NIR and FRET imaging. Bioconjugate Chem. 2012, 23, 981–992.

    Article  Google Scholar 

  28. [28]

    Luo, J. D.; Xie, Z. L.; Lam, J. W. Y.; Cheng, L.; Chen, H. Y.; Qiu, C. F.; Kwok, H. S.; Zhan, X. W.; Liu, Y. Q.; Zhu, D. B. et al. Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. 2001, 1740–1741.

    Google Scholar 

  29. [29]

    Qin, W.; Li, K.; Feng, G. X.; Li, M.; Yang, Z. Y.; Liu, B.; Tang, B. Z. Bright and photostable organic fluorescent dots with aggregation-induced emission characteristics for noninvasive long-term cell imaging. Adv. Funct. Mater. 2014, 24, 635–643.

    Article  Google Scholar 

  30. [30]

    Hong, Y. N.; Lam, J. W. Y.; Tang, B. Z. Aggregationinduced emission: Phenomenon, mechanism and applications. Chem. Commun. 2009, 4332–4353.

    Google Scholar 

  31. [31]

    Hu, R.; Yang, C. B.; Wang, Y. C.; Lin, G. M.; Qin, W.; Ouyang, Q. L.; Law, W. C.; Nguyen, Q. T.; Yoon, H.; Wang, X. M. et al. Aggregation-induced emission (AIE) dye loaded polymer nanoparticles for gene silencing in pancreatic cancer and their in vitro and in vivo biocompatibility evaluation. Nano Res. 2015, 8, 1563–1576.

    Article  Google Scholar 

  32. [32]

    Lee, K. J.; Nallathamby, P. D.; Browning, L. M.; Osgood, C. J.; Xu, X. H. N. In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano 2007, 1, 133–143.

    Article  Google Scholar 

  33. [33]

    Wang, Y. L.; Seebald, J. L.; Szeto, D. P.; Irudayaraj, J. Biocompatibility and biodistribution of surface-enhanced Raman scattering nanoprobes in zebrafish embryos: In vivo and multiplex imaging. ACS Nano 2010, 4, 4039–4053.

    Article  Google Scholar 

  34. [34]

    Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J. L.; Liu, R. R.; Liu, J. Z.; Zhang, X. H.; Liu, H. W.; Liu, B. et al. Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing. Sci. Rep. 2013, 3, 1150.

    Google Scholar 

  35. [35]

    Li, K.; Zhao, X.; Zhai, Y.; Chen, G.; Lee, E. H.; He, S. A study on the biocompatibility of surface-modified Au/Ag alloyed nanobox particles in zebrafish in terms of mortality rate, hatch rate and imaging of particle distribution behavior. Prog. Electromagn. Res. 2015, 150, 89–96.

    Article  Google Scholar 

  36. [36]

    Duan, J. C.; Yu, Y. B.; Li, Y.; Yu, Y.; Sun, Z. W. Cardiovascular toxicity evaluation of silica nanoparticles in endothelial cells and zebrafish model. Biomaterials 2013, 34, 5853–5862.

    Article  Google Scholar 

  37. [37]

    Zhai, Y. X.; Zhao, X. Y.; Sheng, J. H.; Gao, X. W.; Ou, Z.; Xu, Z. P. Ribonuclease like 5 regulates zebrafish yolk extension by suppressing a p53-dependent DNA damage response pathway. Int. J. Biochem. Cell. Biol. 2015, 69, 12–19.

    Article  Google Scholar 

  38. [38]

    Qian, J.; Zhu, Z. F.; Qin, A. J.; Qin, W.; Chu, L. L.; Cai, F. H.; Zhang, H. Q.; Wu, Q.; Hu, R. R.; Tang, B. Z. et al. High-order non-linear optical effects in organic luminogens with aggregation-induced emission. Adv. Mater. 2015, 27, 2332–2339.

    Article  Google Scholar 

  39. [39]

    He, G. S.; Tan, L. S.; Zheng, Q. D.; Prasad, P. N. Multiphoton absorbing materials: Molecular designs, characterizations, and applications. Chem. Rev. 2008, 108, 1245–1330.

    Article  Google Scholar 

  40. [40]

    Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995, 203, 253–310.

    Article  Google Scholar 

  41. [41]

    Cheng, H.; Qin, W.; Zhu, Z. F.; Qian, J.; Qin, A.; Tang, B. Z.; He, S. Nanoparticles with aggregation-induced emission for monitoring long time cell membrane interactions. Prog. Electromagn. Res. 2013, 140, 313–325.

    Article  Google Scholar 

Download references

Author information



Corresponding authors

Correspondence to Guangdi Chen, Ben Zhong Tang or Jun Qian.

Additional information

These authors contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, D., Zhao, X., Qin, W. et al. Toxicity assessment and long-term three-photon fluorescence imaging of bright aggregation-induced emission nanodots in zebrafish. Nano Res. 9, 1921–1933 (2016).

Download citation


  • biocompatible organic nanodots
  • aggregation-induced emission
  • three-photon fluorescence
  • bioimaging
  • zebrafish