Non-Linear Optical Microscopy Sheds Light on Cardiovascular Disease

Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue's morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (BSHG) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression.

诸多心脏疾病均伴随纤维化程度升高与原纤维胶原（fibrillar collagen）组织结构重塑。当前临床常规通过侵入式组织学与免疫组化方法评估纤维化程度，但此类手段仅能对组织针对疾病的形态学适应性改变提供有限且定性的认知。本研究拟借助双光子激发显微镜（Two-Photon Excitation Microscopy, TPE）与二次谐波产生（Second Harmonic Signal Generation, SHG）两种非线性光学显微技术，通过对心肌组织内胶原网络的三维成像，实现纤维化程度升高的定量评估。该方法无需对样本进行染色：诸多内源性荧光基团可通过双光子机制被激发，而胶原等高度非中心对称结构可产生较强的二次谐波信号。本研究首次提出采用三维定量分析方法，对心肌梗死后心力衰竭大鼠模型的组织样本中纤维化程度升高情况进行精准评估。研究证实，仅需利用背向二次谐波（backward SHG, BSHG）信号即可实现纤维化变化的检测，这是因为真正的体内应用场景仅能获取背向传播的二次谐波信号。在梗死发生20周后采集的远隔存活心肌组织中，检测到I型胶原（collagen I）纤维化程度升高5倍。同时研究发现纤维化的空间分布发生显著改变，为解析疾病进展的形态学特征提供了新的视角。