Article

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
  • Download Citation
Received:
Accepted:
Published online:

Abstract

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.

  • Subscribe to Nature Biomedical Engineering for full access:

    $99

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Mohler, E. R. et al. Bone formation and inflammation in cardiac valves. Circulation 103, 1522–1528 (2001).

  2. 2.

    Nkomo, V. T. et al. Burden of valvular heart diseases: a population-based study. Lancet 368, 1005–1011 (2006).

  3. 3.

    Freeman, R. V. & Otto, C. M. Spectrum of calcific aortic valve disease: pathogenesis, disease progression, and treatment strategies. Circulation 111, 3316–3326 (2005).

  4. 4.

    Rajamannan, N. M. et al. Calcific aortic valve disease: not simply a degenerative process: a review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Executive summary: Calcific aortic valve disease-2011 update. Circulation 124, 1783–1791 (2011).

  5. 5.

    Sider, K. L., Blaser, M. C. & Simmons, C. A. Animal models of calcific aortic valve disease. Int. J. Inflam. 2011, 364310 (2011).

  6. 6.

    Wexler, L. et al. Coronary artery calcification: pathophysiology, epidemiology, imaging methods, and clinical implications: a statement for health professionals from the American Heart Association. Circulation 94, 1175–1192 (1996).

  7. 7.

    Linefsky, J. & Otto, C. in Cardiovascular Imaging (ed. Aikawa, E.) 225–249 (Springer, Cham, 2015).

  8. 8.

    Fuster, V. et al. Atherothrombosis and high-risk plaque: part II: approaches by noninvasive computed tomographic/magnetic resonance imaging. J. Am. Coll. Cardiol. 46, 1209–1218 (2005).

  9. 9.

    New, S. E. P. & Aikawa, E. Molecular imaging insights into early inflammatory stages of arterial and aortic valve calcification. Circ. Res. 108, 1381–1391 (2011).

  10. 10.

    Konig, K., Schenke-Layland, K., Riemann, I. & Stock, U. A. Multiphoton autofluorescence imaging of intratissue elastic fibers. Biomaterials 26, 495–500 (2005).

  11. 11.

    Alavi, S. H., Ruiz, V., Krasieva, T., Botvinick, E. L. & Kheradvar, A. Characterizing the collagen fiber orientation in pericardial leaflets under mechanical loading conditions. Ann. Biomed. Eng. 41, 547–561 (2013).

  12. 12.

    Gerson, C. J., Goldstein, S. & Heacox, A. E. Retained structural integrity of collagen and elastin within cryopreserved human heart valve tissue as detected by two-photon laser scanning confocal microscopy. Cryobiology 59, 171–179 (2009).

  13. 13.

    Dweck, M. et al. 18F-sodium fluoride is a marker of active calcification and disease progression in patients with aortic stenosis. Circ. Cardiovasc. Imaging 7, 371–378 (2014).

  14. 14.

    Dweck, M. R. et al. Assessment of valvular calcification and inflammation by positron emission tomography in patients with aortic stenosis. Circulation 125, 76–86 (2012).

  15. 15.

    Jenkins, W. S. et al. Valvular 18F-fluoride and 18F-fluorodeoxyglucose uptake predict disease progression and clinical outcome in patients with aortic stenosis. J. Am. Coll. Cardiol. 66, 1200–1201 (2015).

  16. 16.

    Vesey, A., Dweck, M. & Newby, D. in Cardiovascular Imaging (ed. Aikawa, E.) 201–223 (Springer, Cham, 2015).

  17. 17.

    Ballyns, J. J. & Bonassar, L. J. Image‐guided tissue engineering. J. Cell. Mol. Med. 13, 1428–1436 (2009).

  18. 18.

    Feuchtner, G. Imaging of cardiac valves by computed tomography. Scientifica 2013, 270579 (2013).

  19. 19.

    Goo, H. W. CT radiation dose optimization and estimation: an update for radiologists. Korean J. Radiol. 13, 1–11 (2012).

  20. 20.

    Hjortnaes, J. et al. Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: a role for inflammation. Eur. Heart J. 31, 1975–1984 (2010).

  21. 21.

    Jaffer, F. A., Libby, P. & Weissleder, R. Optical and multimodality molecular imaging: insights into atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 29, 1017–1024 (2009).

  22. 22.

    Kherlopian, A. et al. A review of imaging techniques for systems biology. BMC Syst. Biol. 2, 74 (2008).

  23. 23.

    Hutson, H. N. et al. Calcific aortic valve disease is associated with layer-specific alterations in collagen architecture. PLoS ONE 11, e0163858 (2016).

  24. 24.

    Bertazzo, S. et al. Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification. Nat. Mater. 12, 576–583 (2013).

  25. 25.

    Williams, R. M., Zipfel, W. R. & Webb, W. W. Multiphoton microscopy in biological research. Curr. Opin. Chem. Biol. 5, 603–608 (2001).

  26. 26.

    Georgakoudi, I. & Quinn, K. P. Optical imaging using endogenous contrast to assess metabolic state. Annu. Rev. Biomed. Eng. 14, 351–367 (2012).

  27. 27.

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

  28. 28.

    Zipfel, W. R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl Acad. Sci. USA 100, 7075–7080 (2003).

  29. 29.

    Cui, J. Z. et al. Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy. J. Struct. Biol. 187, 242–253 (2014).

  30. 30.

    König, K., Schenke-Layland, K., Riemann, I. & Stock, U. Multiphoton autofluorescence imaging of intratissue elastic fibers. Biomaterials 26, 495–500 (2005).

  31. 31.

    Fitzmaurice, M. et al. Argon ion laser-excited autofluorescence in normal and atherosclerotic aorta and coronary arteries: morphologic studies. Am. Heart J. 118, 1028–1038 (1989).

  32. 32.

    Benninger, R. K. & Piston, D. W. Two‐photon excitation microscopy for the study of living cells and tissues. Curr. Protoc. Cell Biol. 4, 4.1124 (2013).

  33. 33.

    Huang, S., Heikal, A. A. & Webb, W. W. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys. J. 82, 2811–2825 (2002).

  34. 34.

    Croce, A. & Bottiroli, G. Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur. J. Histochem. 58, 2461 (2014).

  35. 35.

    Perry, S. W., Burke, R. M. & Brown, E. B. Two-photon and second harmonic microscopy in clinical and translational cancer research. Ann. Biomed. Eng. 40, 277–291 (2012).

  36. 36.

    Chen, X., Nadiarynkh, O., Plotnikov, S. & Campagnola, P. J. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat. Protoc. 7, 654–669 (2012).

  37. 37.

    Cicchi, R. et al. Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J. Biophotonics 3, 34–43 (2010).

  38. 38.

    Williams, R. M., Zipfel, W. R. & Webb, W. W. Interpreting second-harmonic generation images of collagen I fibrils. Biophys. J. 88, 1377–1386 (2005).

  39. 39.

    Campagnola, P. J. & Loew, L. M. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat. Biotechnol. 21, 1356–1360 (2003).

  40. 40.

    Liu, Z. et al. Rapid three-dimensional quantification of voxel-wise collagen fiber orientation. Biomed. Opt. Express 6, 2294–2310 (2015).

  41. 41.

    Liu, Z. et al. Automated quantification of three-dimensional organization of fiber-like structures in biological tissues. Biomaterials 116, 34–47 (2017).

  42. 42.

    Zoumi, A., Yeh, A. & Tromberg, B. J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl Acad. Sci. USA 99, 11014–11019 (2002).

  43. 43.

    Richards-Kortum, R. & Sevick-Muraca, E. Quantitative optical spectroscopy for tissue diagnosis. Annu. Rev. Phys. Chem. 47, 555–606 (1996).

  44. 44.

    Cho, A., Suzuki, S., Hatakeyama, J., Haruyama, N. & Kulkarni, A. B. A method for rapid demineralization of teeth and bones. Open Dent. J. 4, 223–229 (2010).

  45. 45.

    Berzina-Cimdina, L. & Borodajenko, N. in Infrared Spectroscopy—Materials Science, Engineering and Technology (ed. Theophile, T.) Ch. 6 (InTech, Rijeka, 2012).

  46. 46.

    Ko, A. C. T. et al. Multimodal nonlinear optical imaging of atherosclerotic plaque development in myocardial infarction-prone rabbits. J. Biomed. Opt. 15, 020501 (2010).

  47. 47.

    Verbunt, R. J. A. M. et al. Characterization of ultraviolet laser-induced autofluorescence of ceroid deposits and other structures in atherosclerotic plaques as a potential diagnostic for laser angiosurgery. Am. Heart J. 123, 208–216 (1992).

  48. 48.

    Le, T. T., Langohr, I. M., Locker, M. J., Sturek, M. & Cheng, J.-X. Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy. J. Biomed. Opt.  12, 054007 (2007).

  49. 49.

    Wang, Y.-L. & Pelham R. J. Jr, in Methods in Enzymology Vol 298 (ed. Richard, B. V.) 489–496 (Academic, Cambridge, MA, 1998).

  50. 50.

    O’Brien, K. D. Pathogenesis of calcific aortic valve disease: a disease process comes of age (and a good deal more). Arterioscler. Thromb. Vasc. Biol. 26, 1721–1728 (2006).

  51. 51.

    Evans, C. L. & Xie, X. S. Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu. Rev. Anal. Chem. 1, 883–909 (2008).

  52. 52.

    Sider, K. L., Blaser, M. C. & Simmons, C. A. Animal models of calcific aortic valve disease. Int. J. Inflam. 2011, 364310 (2011).

  53. 53.

    Tanaka, K. et al. Age-associated aortic stenosis in apolipoprotein E-deficient mice. J. Am. Coll. Cardiol. 46, 134–141 (2005).

  54. 54.

    Hutcheson, J. D., Aikawa, E. & Merryman, W. D. Potential drug targets for calcific aortic valve disease. Nat. Rev. Cardiol. 11, 218–231 (2014).

  55. 55.

    Aikawa, E. et al. Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation 116, 2841–2850 (2007).

  56. 56.

    Giachelli, C. M. Ectopic calcification. Am. J. Pathol. 154, 671–675 (1999).

  57. 57.

    Richards‐Kortum, R. et al. Spectroscopic diagnosis of colonic dysplasia. Photochem. Photobiol. 53, 777–786 (1991).

  58. 58.

    Guerraty, M. & Mohler, E. R. Models of aortic valve calcification. J. Invest. Med. 55, 278–283 (2007).

  59. 59.

    Hutcheson, J. D. et al. Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques. Nat. Mater. 15, 335–343 (2016).

  60. 60.

    Clark, C. R., Bowler, M. A., Snider, J. C. & Merryman, W. D. Targeting cadherin-11 prevents Notch1-mediated calcific aortic valve disease. Circulation 135, 2448–2450 (2017).

  61. 61.

    Perosky, J. E. et al. Early detection of heterotopic ossification using near‐infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury. J. Orthopaed. Res. 32, 1416–1423 (2014).

  62. 62.

    Zhang, Y. et al. A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy. Proc. Natl Acad. Sci. USA 109, 12878–12883 (2012).

  63. 63.

    Murari, K. et al. Compensation-free, all-fiber-optic, two-photon endomicroscopy at 1.55 μm. Opt. Lett. 36, 1299–1301 (2011).

  64. 64.

    Nishimura, R. et al. 2014 AHA/ACC guideline for the management of patients with valvular heart disease: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation 129, 2440–2492 (2014).

  65. 65.

    Mathieu, P. et al. The pathology and pathobiology of bicuspid aortic valve: state of the art and novel research perspectives. J. Pathol. Clin. Res. 1, 195–206 (2015).

  66. 66.

    Sethuraman, S., Amirian, J. H., Litovsky, S. H., Smalling, R. W. & Emelianov, S. Y. Spectroscopic intravascular photoacoustic imaging to differentiate atherosclerotic plaques. Opt. Express 16, 3362–3367 (2008).

  67. 67.

    Wang, B. et al. Intravascular photoacoustic imaging. IEEE J. Quantum Electron. 16, 588–599 (2010).

  68. 68.

    Fu, H. L. et al. Flexible miniature compound lens design for high-resolution optical coherence tomography balloon imaging catheter. J. Biomed. Opt. 13, 060502 (2008).

  69. 69.

    Hassan, T., Piton, N., Lachkar, S., Salaun, M. & Thiberville, L. A novel method for in vivo imaging of solitary lung nodules using navigational bronchoscopy and confocal laser microendoscopy. Lung 193, 773–778 (2015).

  70. 70.

    Achenbach, S. et al. Detection of calcified and noncalcified coronary atherosclerotic plaque by contrast-enhanced, submillimeter multidetector spiral computed tomography: a segment-based comparison with intravascular ultrasound. Circulation 109, 14–17 (2004).

  71. 71.

    Radi, M. J. Calcium oxalate crystals in breast biopsies. An overlooked form of microcalcification associated with benign breast disease. Arch. Pathol. Lab. Med. 113, 1367–1369 (1989).

  72. 72.

    Sullivan, K., Quinn, K., Tang, K., Georgakoudi, I. & Black, L. III Extracellular matrix remodeling following myocardial infarction influences the therapeutic potential of mesenchymal stem cells. Stem Cell Res. Ther. 5, 1–16 (2014).

  73. 73.

    Quinlan, A. M. & Billiar, K. L. Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation. J. Biomed. Mater. Res. A 100, 2474–2482 (2012).

  74. 74.

    Liao, P.-S., Chen, T.-S. & Chung, P.-C. A fast algorithm for multilevel thresholding. J. Inf. Sci. Eng. 17, 713–727 (2001).

  75. 75.

    Levitt, J. M. et al. Diagnostic cellular organization features extracted from autofluorescence images. Opt. Lett. 32, 3305–3307 (2007).

  76. 76.

    Xylas, J., Alt-Holland, A., Garlick, J., Hunter, M. & Georgakoudi, I. Intrinsic optical biomarkers associated with the invasive potential of tumor cells in engineered tissue models. Biomed. Opt. Express 1, 1387–1400 (2010).

  77. 77.

    Xylas, J. et al. Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development. Int. J. Cancer 136, 322–332 (2015).

  78. 78.

    Pouli, D. et al. Imaging mitochondrial dynamics in human skin reveals depth-dependent hypoxia and malignant potential for diagnosis. Sci. Transl. Med. 8, 367ra169 (2016).

  79. 79.

    Zeadin, M. et al. Effect of leptin on vascular calcification in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 29, 2069–2075 (2009).

Download references

Acknowledgements

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

Affiliations

  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

Authors

  1. Search for Lauren M. Baugh in:

  2. Search for Zhiyi Liu in:

  3. Search for Kyle P. Quinn in:

  4. Search for Sam Osseiran in:

  5. Search for Conor L. Evans in:

  6. Search for Gordon S. Huggins in:

  7. Search for Philip W. Hinds in:

  8. Search for Lauren D. Black III in:

  9. Search for Irene Georgakoudi in:

Contributions

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)