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

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.