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

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.

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Fig. 1: Visual analysis of TPEF images compared with Alizarin Red staining of a human CAVD nodule, a rat bone and a PAAM-gel-grown nodule.
Fig. 2: Representative images of human CAVD nodules, rat bone samples and PAAM gel nodules taken using SEM and TPEF.
Fig. 3: Effect of EDTA treatment on endogenous TPEF.
Fig. 4: Analysis of TPEF emission spectra reveals the emission spectrum of MAF.
Fig. 5: MAF images enable enhanced visualization of mineralized sample regions.
Fig. 6: MAF signal overlaps with CARS images.
Fig. 7: An ApoE–/– mouse model confirms the use of MAF as a quantifiable metric for calcification.
Fig. 8: Time-lapse experiment of calcification on PAAM gels shows measured change in nodule size.

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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.

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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.

Corresponding authors

Correspondence to Lauren D. Black III or Irene Georgakoudi.

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Supplementary Video 1

Representative image stack of a human CAVD valve

Supplementary Video 2

Representative image stack of an EDTA-treated human CAVD valve

Supplementary Video 3

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

Supplementary Video 4

Representative image stack of a rat bone

Supplementary Video 5

Representative image stack of an EDTA-treated rat bone

Supplementary Video 6

Representative image stack of a nodule grown on a PAAM gel

Supplementary Video 7

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

Supplementary Video 8

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

Supplementary Video 9

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

Supplementary Video 10

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

Supplementary Video 11

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

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Baugh, L.M., Liu, Z., Quinn, K.P. et al. Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease. Nat Biomed Eng 1, 914–924 (2017). https://doi.org/10.1038/s41551-017-0152-3

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