Nano Research

, Volume 8, Issue 9, pp 3027–3034 | Cite as

Biological imaging without autofluorescence in the second near-infrared region

  • Shuo Diao
  • Guosong Hong
  • Alexander L. Antaris
  • Jeffrey L. Blackburn
  • Kai Cheng
  • Zhen Cheng
  • Hongjie DaiEmail author
Research Article


Fluorescence imaging is capable of acquiring anatomical and functional information with high spatial and temporal resolution. This imaging technique has been indispensable in biological research and disease detection/diagnosis. Imaging in the visible and to a lesser degree, in the near-infrared (NIR) regions below 900 nm, suffers from autofluorescence arising from endogenous fluorescent molecules in biological tissues. This autofluorescence interferes with fluorescent molecules of interest, causing a high background and low detection sensitivity. Here, we report that fluorescence imaging in the 1,500–1,700-nm region (termed “NIR-IIb”) under 808-nm excitation results in nearly zero tissue autofluorescence, allowing for background-free imaging of fluorescent species in otherwise notoriously autofluorescent biological tissues, including liver. Imaging of the intrinsic fluorescence of individual fluorophores, such as a single carbon nanotube, can be readily achieved with high sensitivity and without autofluorescence background in mouse liver within the 1,500–1,700-nm wavelength region.


fluorescence imaging second near-infrared nanotechnology autofluorescence 


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  1. [1]
    Ntziachristos, V.; Chance, B. Breast imaging technology: Probing physiology and molecular function using optical imaging-applications to breast cancer. Breast Cancer Res. 2001, 3, 41–46.CrossRefGoogle Scholar
  2. [2]
    He, X. X.; Gao, J. H.; Gambhir, S. S.; Cheng, Z. Nearinfrared fluorescent nanoprobes for cancer molecular imaging: Status and challenges. Trends Mol. Med. 2010, 16, 574–583.CrossRefGoogle Scholar
  3. [3]
    Shang, L.; Dörlich, R. M.; Trouillet, V.; Bruns, M.; Nienhaus, G. U. Ultrasmall fluorescent silver nanoclusters: Protein adsorption and its effects on cellular responses. Nano Res. 2012, 5, 531–542.CrossRefGoogle Scholar
  4. [4]
    Le Guével, X.; Spies, C.; Daum, N.; Jung, G.; Schneider, M. Highly fluorescent silver nanoclusters stabilized by glutathione: A promising fluorescent label for bioimaging. Nano Res. 2012, 5, 379–387.CrossRefGoogle Scholar
  5. [5]
    Guo, W. S.; Yang, W. T.; Wang, Y.; Sun, X. L.; Liu, Z. Y.; Zhang, B. B.; Chang, J.; Chen, X.Y. Color-tunable Gd-Zn- Cu-In-S/ZnS quantum dots for dual modality magnetic resonance and fluorescence imaging. Nano Res. 2014, 7, 1581–1591.CrossRefGoogle Scholar
  6. [6]
    Sevick-Muraca, E. M. Translation of near-infrared fluorescence imaging technologies: Emerging clinical applications. Annu. Rev. Med. 2012, 63, 217–231.CrossRefGoogle Scholar
  7. [7]
    Baschong, W.; Suetterlin, R.; Laeng, R. H. Control of autofluorescence of archival formaldehyde-fixed, paraffinembedded tissue in confocal laser scanning microscopy (CLSM). J. Histochem. Cytochem. 2001, 49, 1565–1571.CrossRefGoogle Scholar
  8. [8]
    Niku, M.; Pessa-Morikawa, T.; Taponen, J.; Iivanainen, A. Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle. BMC Vet. Res. 2007, 3, 29.Google Scholar
  9. [9]
    Sun, Y.; Yu, H.; Zheng, D.; Cao, Q.; Wang, Y.; Harris, D.; Wang, Y. P. Sudan black B reduces autofluorescence in murine renal tissue. Arch. Pathol. Lab. Med. 2011, 135, 1335–1342.CrossRefGoogle Scholar
  10. [10]
    Potter, K. A.; Simon, J. S.; Velagapudi, B.; Capadona, J. R. Reduction of autofluorescence at the microelectrode–cortical tissue interface improves antibody detection. J. Neurosci. Methods 2012, 203, 96–105.CrossRefGoogle Scholar
  11. [11]
    Schnell, S. A.; Staines, W. A.; Wessendorf, M. W. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J. Histochem. Cytochem. 1999, 47, 719–730.CrossRefGoogle Scholar
  12. [12]
    Masella, B. D.; Williams, D. R.; Fischer, W.; Rossi, E. A.; Hunter, J. J. Long-term reduction in infrared autofluorescence caused by infrared light below the maximum permissible exposure. Invest. Ophthalmol. Visual Sci. 2014, IOVS-13-12562.Google Scholar
  13. [13]
    Neumann, M.; Gabel, D. Simple method for reduction of autofluorescence in fluorescence microscopy. J. Histochem. Cytochem. 2002, 50, 437–439.CrossRefGoogle Scholar
  14. [14]
    Viegas, M. S.; Martins, T. C.; Seco, F.; Do Carmo, A. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. Eur. J. Histochem. 2007, 51, 59–66.Google Scholar
  15. [15]
    Diao, S.; Hong, G. S.; Robinson, J. T.; Jiao, L. Y.; Antaris, A. L.; Wu, J. Z.; Choi, C. L.; Dai, H. J. Chirality enriched (12, 1) and (11, 3) single-walled carbon nanotubes for biological imaging. J. Am. Chem. Soc. 2012, 134, 16971–16974.CrossRefGoogle Scholar
  16. [16]
    Hong, G. S.; Diao, S.; Chang, J. L.; Antaris, A. L.; Chen, C. X.; Zhang, B.; Zhao, S.; Atochin, D. N.; Huang, P. L.; Andreasson, K. I. et al. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nat. Photonics 2014. 8, 723–730.CrossRefGoogle Scholar
  17. [17]
    Hong, G. S.; Robinson, J. T.; Zhang, Y. J.; Diao, S.; Antaris, A. L.; Wang, Q. B.; Dai, H. J. In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angew. Chem. Int. Ed. 2012, 51, 9818–9821.CrossRefGoogle Scholar
  18. [18]
    Hong, G. S.; Zou, Y. P.; Antaris, A. L.; Diao, S.; Wu, D.; Cheng, K.; Zhang, X. D.; Chen, C. X.; Liu, B.; He, Y. H. et al. Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window. Nat. Commun. 2014, 5. 4206.Google Scholar
  19. [19]
    Naczynski, D. J.; Tan, M. C.; Zevon, M.; Wall, B.; Kohl, J.; Kulesa, A.; Chen, S.; Roth, C. M.; Riman, R. E.; Moghe, P. V. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 2013, 4. 2199.Google Scholar
  20. [20]
    Tsukasaki, Y.; Morimatsu, M.; Nishimura, G.; Sakata, T.; Yasuda, H.; Komatsuzaki, A.; Watanabe, T. M.; Jin, T. Synthesis and optical properties of emission-tunable PbS/CdS core–shell quantum dots for in vivo fluorescence imaging in the second near-infrared window. RSC Adv. 2014, 4, 41164–41171.CrossRefGoogle Scholar
  21. [21]
    Yi, H. J.; Ghosh, D.; Ham, M.-H.; Qi, J. F.; Barone, P. W.; Strano, M. S.; Belcher, A. M. M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett. 2012, 12, 1176–1183.CrossRefGoogle Scholar
  22. [22]
    Tao, Z. M.; Hong, G. S.; Shinji, C.; Chen, C. X.; Diao, S.; Antaris, A. L.; Zhang, B.; Zou, Y. P.; Dai, H. J. Biological imaging using nanoparticles of small organic molecules with fluorescence emission at wavelengths longer than 1,000 nm. Angew. Chem. Int. Ed. 2013. 52, 13002–13006.CrossRefGoogle Scholar
  23. [23]
    Bashkatov, A. N.; Genina, E. A.; Kochubey, V. I.; Tuchin, V. V. Optical properties of human skin, subcutaneous and mucous tissues in the wavelength range from 400 to 2,000 nm. J. Phys. D: Appl. Phys. 2005, 38, 2543–2555.CrossRefGoogle Scholar
  24. [24]
    Gu, L.; Hall, D. J.; Qin, Z. T.; Anglin, E.; Joo, J.; Mooney, D. J.; Howell, S. B.; Sailor, M. J. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles. Nat. Commun. 2013, 4, 3326.Google Scholar
  25. [25]
    Diao, S.; Blackburn, J. L.; Hong, G. S.; Antaris, A. L.; Chang, J. L.; Wu, J. Z.; Zhang, B.; Kuo, C. J.; Dai, H. J. Fluorescence imaging in vivo up to 1,700 nm. arXiv preprint arXiv:1502.02775 2015.Google Scholar
  26. [26]
    Priceton Instruments. 2D-OMA V InGaAs camera user manual.Google Scholar
  27. [27]
    Villa, I.; Vedda, A.; Cantarelli, I. X.; Pedroni, M.; Piccinelli, F.; Bettinelli, M.; Speghini, A.; Quintanilla, M.; Vetrone, F.; Rocha, U. et al. 1.3 µm emitting SrF2: Nd3+ nanoparticles for high contrast in vivo imaging in the second biological window. Nano Res. 2015, 8, 1–17.CrossRefGoogle Scholar
  28. [28]
    Pukelsheim, F. The three sigma rule. Amer. Statist. 1994, 48, 88–91.Google Scholar
  29. [29]
    Abdo, Z.; Schüette, U. M. E.; Bent, S. J.; Williams, C. J.; Forney, L. J.; Joyce, P. Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes. Environ. Microbiol. 2006, 8, 929–938.CrossRefGoogle Scholar
  30. [30]
    Shrivastava, A.; Gupta, V. B. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron. Young Sci. 2011, 2, 21–25.CrossRefGoogle Scholar
  31. [31]
    Dill, K.; Montgomery, D. D.; Ghindilis, A. L.; Schwarzkopf, K. R.; Ragsdale, S. R.; Oleinikov, A. V. Immunoassays based on electrochemical detection using microelectrode arrays. Biosens. Bioelectron. 2004, 20, 736–742.CrossRefGoogle Scholar
  32. [32]
    Hatami, S.; Würth, C.; Kaiser, M.; Leubner, S.; Gabriel, S.; Bahrig, L.; Lesnyak, V.; Pauli, J.; Gaponik, N.; Eychmüller, A. et al. Absolute photoluminescence quantum yields of IR26 and IR-emissive Cd1–x Hgx Te and PbS quantum dots–methodand material-inherent challenges. Nanoscale 2015, 7, 133–143.CrossRefGoogle Scholar
  33. [33]
    Welsher, K.; Liu, Z.; Sherlock, S. P.; Robinson, J. T.; Chen, Z.; Daranciang, D.; Dai, H. J. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat. Nanotechnol. 2009, 4, 773–780.CrossRefGoogle Scholar
  34. [34]
    Mistry, K. S.; Larsen, B. A.; Blackburn, J. L. High-yield dispersions of large-diameter semiconducting single-walled carbon nanotubes with tunable narrow chirality distributions. ACS nano 2013, 7, 2231–2239.CrossRefGoogle Scholar
  35. [35]
    Liu, Z.; Davis, C.; Cai, W. B.; He, L. N.; Chen, X. Y.; Dai, H. J. Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy. Proc. Natl. Acad. Sci. 2008, 105, 1410–1415.CrossRefGoogle Scholar
  36. [36]
    Hong, G. S.; Wu, J. Z.; Robinson, J. T.; Wang, H. L.; Zhang, B.; Dai, H. J. Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates. Nat. Commun. 2012, 3, 700.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Shuo Diao
    • 1
  • Guosong Hong
    • 1
  • Alexander L. Antaris
    • 1
  • Jeffrey L. Blackburn
    • 2
  • Kai Cheng
    • 3
  • Zhen Cheng
    • 3
  • Hongjie Dai
    • 1
    Email author
  1. 1.Department of ChemistryStanford UniversityStanfordUSA
  2. 2.Chemical and Materials Science CenterNational Renewable Energy LaboratoryColoradoUSA
  3. 3.Molecular Imaging Program at Stanford (MIPS) and Department of RadiologyStanford UniversityStanfordUSA

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