Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.

相干抗斯托克斯拉曼散射（CARS）和二次谐波产生（SHG）是非线性技术，可对细胞和组织进行无标记，无损和无创性成像。尽管之前已经进行过实时成像研究，但仍未解决有关它们不会在敏感生物样品的分子水平上引起任何变化的担忧。如果将CARS / SHG显微镜用作评估正在发育的新组织的非侵入性，无标签工具，则这对于干细胞分化和组织工程尤为重要。在这项工作中，我们使用CARS和SHG显微镜在三维培养物中监测了人类胎儿-股骨来源的骨骼细胞向软骨的分化，并证明了随着时间的推移，同一发育中的新组织的实时成像。我们的工作最终确定，非线性无标记成像不会在分化的不同阶段改变表型或基因表达，并且不会对人体骨骼细胞的生长和行为产生不利影响。此外，我们显示，CARS显微镜可以在生物工程化的新软骨中成像不同的目标分子，包括脂质，蛋白质和糖胺聚糖。这些研究证明了实时CARS和SHG成像的无标签，真正无创性质，以及它们在骨骼研究，再生医学和组织工程中的价值和翻译潜力。我们显示，CARS显微镜可以在生物工程化的新软骨中成像不同的目标分子，包括脂质，蛋白质和糖胺聚糖。这些研究证明了实时CARS和SHG成像的无标签，真正无创性质，以及它们在骨骼研究，再生医学和组织工程中的价值和翻译潜力。我们显示，CARS显微镜可以在生物工程化的新软骨中成像不同的目标分子，包括脂质，蛋白质和糖胺聚糖。这些研究证明了实时CARS和SHG成像的无标签，真正无创性质，以及它们在骨骼研究，再生医学和组织工程中的价值和翻译潜力。