CARS和SHG显微镜检查，以跟踪纤维蛋白凝胶3D培养物中活的人角膜成纤维细胞和间充质干细胞中的胶原蛋白产生
CARS and SHG microscopy to follow the collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin gel 3D cultures
摘要：
相干反斯托克斯拉曼散射（CARS）显微镜与二次谐波发生（SHG）技术相结合，以跟踪3D支架内干细胞分化的早期阶段。CARS显微镜可以检测活细胞中的脂质膜和液滴隔室，SHG显微镜可以为具有非中心对称有序结构的分子（如胶原蛋白）提供强大的成像对比度。hMSC分化的第一个证据之一是细胞外基质（ECM）的形成，其中胶原蛋白是其主要成分。这项工作证明了多模式CARS和SHG显微镜作为一种强大的非侵入性无标记技术，可用于研究活细胞3D培养中的胶原蛋白生产动态。其在长期（4周）实验中对细胞形态和产生的胶原分布成像的能力允许获得关于细胞-支架相互作用和ECM产生的重要信息。在检测胶原蛋白中达到的非常低的极限已经允许绘制甚至由细胞在几个小时的培养中产生的少量胶原蛋白。这表明多模式CARS和SHG显微镜作为一种新的方法来跟踪细胞胶原蛋白的产生和细胞分化过程。此外，实验表明，该技术是一个强大的工具，非常厚的部分（约4毫米）的成像。对纤维蛋白凝胶培养物中的间充质干细胞进行的研究证实了支架诱导分化刺激。以非破坏性的方式监测支架内干细胞的分化将是再生医学和组织工程领域的一个重要优势。
英文摘要：
Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold. CARS microscopy can detect lipid membranes and droplet compartments in living cells and SHG microscopy enables a strong imaging contrast for molecules with a non-centrosymmetric ordered structure like collagen. One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component. This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to investigate the collagen production dynamic in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permitted to map even the small amount of collagen produced by the cells in few hours of culture. This demonstrates multimodal CARS and SHG microscopy as a novel method to follow cells collagen production and cells differentiation process. In addition the experiment shows that the technique is a powerful tool for imaging of very thick sections (about 4 mm). The study conducted on mesenchymal stem cell in fibrin gel cultures confirmed that differentiation stimulus is induced by the scaffold. The monitoring of stem cell differentiation within a scaffold in a non-destructive way will be an important advantage in regenerative medicine and tissue engineering field.
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