Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease

  • Nature Biomedical Engineering 1914924 (2017)
  • doi:10.1038/s41551-017-0152-3
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Calcifications occur during the development of healthy bone and at the onset of calcific aortic-valve disease (CAVD) and many other pathologies. Although the mechanisms regulating early calcium deposition are not fully understood, they may provide targets for new treatments and early interventions. Here, we show that two-photon excited fluorescence (TPEF) can provide quantitative and sensitive readouts of calcific nodule formation, in particular in the context of CAVD. Specifically, by means of the decomposition of TPEF spectral images from excised human CAVD valves and rat bone before and after demineralization, as well as from calcific nodules formed within engineered gels, we identified an endogenous fluorophore that correlates with the level of mineralization in the samples. We then developed a ratiometric imaging approach that provides a quantitative readout of the presence of mineral deposits in early calcifications. TPEF should enable non-destructive, high-resolution imaging of three-dimensional tissue specimens for the assessment of the presence of calcification.

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We are grateful to M. Freytsis for help with collecting the human CAVD valve samples at Tufts Medical Center. We also thank the Jaffe Laboratory for the generous donation of freshly isolated ApoE–/– and wild-type mouse hearts. Financial support was provided by the National Institutes of Health–National Institute of Biomedical Imaging and Bioengineering (awards K99EB017723 and R00EB017723 to K.P.Q., R01HL114794 to G.S.H., P.W.H. and L.D.B., and R01EB007542 to I.G) and American Cancer Society Research Scholar Grant RSG-09-174-01-CCE to I.G.

Author information


  1. Department of Biomedical Engineering, Tufts University, Medford, MA, 02155, USA

    • Lauren M. Baugh
    • , Zhiyi Liu
    • , Kyle P. Quinn
    • , Lauren D. Black III
    •  & Irene Georgakoudi
  2. Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, 72701, USA

    • Kyle P. Quinn
  3. Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, 02129, USA

    • Sam Osseiran
    •  & Conor L. Evans
  4. Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    • Sam Osseiran
  5. Molecular Cardiology Research Center, Tufts Medical Center and Tufts University Sackler School for Graduate Biomedical Sciences, Boston, MA, 02111, USA

    • Gordon S. Huggins
  6. Department of Developmental, Molecular, and Chemical Biology and Program in Genetics, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA, 02111, USA

    • Philip W. Hinds
    •  & Lauren D. Black III


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L.M.B. contributed to conception and design of the experiments, collection, assembly, analysis and interpretation of data, and writing and final approval of the paper. K.P.Q. performed data analysis and interpretation, and contributed to writing and final approval of the paper. Z.L. developed the computational model to quantitatively extract the component contributions from the images acquired at two emission bands and performed the corresponding calculations for both the human CAVD valves and the mouse model valves. G.S.H. and P.W.H. contributed to data interpretation and final approval of the paper. S.O. and C.L.E. performed CARS and TPEF imaging, as well as data interpretation of human CAVD and rat bone samples. G.S.H. also provided the human CAVD valve samples. I.G. directed the image acquisition and image analysis aspects of the study. L.D.B and I.G. contributed to the project conception and design, manuscript writing, data interpretation and final approval of the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Lauren D. Black III or Irene Georgakoudi.

Electronic supplementary material

  1. Supplementary Information

    Supplementary figures and video legends

  2. Life sciences reporting summary

  3. Supplementary Video 1

    Representative image stack of a human CAVD valve

  4. Supplementary Video 2

    Representative image stack of an EDTA-treated human CAVD valve

  5. Supplementary Video 3

    Representative image stack of SHG and TPEF for a human CAVD valve

  6. Supplementary Video 4

    Representative image stack of a rat bone

  7. Supplementary Video 5

    Representative image stack of an EDTA-treated rat bone

  8. Supplementary Video 6

    Representative image stack of a nodule grown on a PAAM gel

  9. Supplementary Video 7

    120-hour time lapse of a PAAM gel seeded with VICs with images of the mineralization taken every 8 hours

  10. Supplementary Video 8

    Cropped time lapse of calculated MAF images of nodules (ROI 1)

  11. Supplementary Video 9

    Cropped time lapse of calculated MAF images of nodules (ROI 2)

  12. Supplementary Video 10

    Cropped time lapse of calculated MAF images of nodules (ROI 3)

  13. Supplementary Video 11

    Cropped time lapse of calculated MAF images of nodules (ROI 4)