Institute for Life Sciences and Department of Chemistry, Highfield Campus, University of Southampton, SO17 1BJ, Southampton, UK.
Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, Faculty of Medicine, University of Southampton, SO16 6YD, Southampton, UK.
Sci Rep. 2019 Apr 3;9(1):5561. doi: 10.1038/s41598-019-41466-w.
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.
相干反斯托克斯拉曼散射（CARS）和二次谐波产生（SHG）是非线性技术，可用于对细胞和组织进行无标记、无损和非侵入式成像分析。虽然之前已经进行了活细胞成像研究，但人们仍然担心这些技术不会在敏感的生物样本中引起任何分子水平的变化。这一点尤其重要，因为如果 CARS/SHG 显微镜要作为评估新组织发育的非侵入性、无标记工具，那么在干细胞分化和组织工程中就需要考虑到这一点。在这项工作中，我们使用 CARS 和 SHG 显微镜监测了三维培养的人胎儿股骨衍生的骨骼细胞向软骨的分化，并证明了同一发育中的新组织随时间的活细胞成像。我们的工作明确证实，非线性无标记成像不会改变分化不同阶段的表型或基因表达，并且对人骨骼细胞的生长和行为没有不良影响。此外，我们还表明 CARS 显微镜允许对生物工程新软骨中的不同感兴趣分子（包括脂质、蛋白质和糖胺聚糖）进行成像。这些研究证明了活细胞 CARS 和 SHG 成像的无标记和真正非侵入性特性，以及它们在骨骼研究、再生医学和组织工程中的价值和转化潜力。
