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.

钙化发生在健康骨骼的发育过程中，以及钙化主动脉瓣疾病（CAVD）和许多其他病理的发作期间。尽管尚不能完全了解调节早期钙沉积的机制，但它们可能为新疗法和早期干预提供目标。在这里，我们显示了双光子激发荧光（TPEF）可以提供钙化结节形成的定量和敏感读数，尤其是在CAVD的情况下。具体而言，通过脱矿前后人CAVD瓣膜和大鼠骨骼的TPEF光谱图像的分解以及工程凝胶中形成的钙化结节的分解，我们确定了与样品矿化水平相关的内源性荧光团。然后，我们开发了一种比例成像方法，该方法可以定量读取早期钙化过程中矿床的存在。TPEF应该能够对三维组织标本进行无损高分辨率成像，以评估钙化的存在。