Chalmers University of Technology, Molecular Microscopy, Department of Chemical and Biological Engineering, SE-412 96 Göteborg, Sweden.
J Biomed Opt. 2011 Feb;16(2):021115. doi: 10.1117/1.3534782.
The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH(2)-groups at 2845 cm(-1) permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.
活的、人类平滑肌细胞在生物合成的纤维素支架中的整合通过非线性显微镜进行监测，目的是构建可收缩的人工血管。结合相干反斯托克斯拉曼散射（CARS）和二次谐波产生（SHG）显微镜，研究了细胞与生物聚合物网络的相互作用。CARS 显微镜探测到 2845 cm(-1)处的 CH(2)-基团，允许对富含脂质的细胞内结构具有高对比度的细胞进行三维成像。SHG 显微镜可视化纤维素支架的纤维，以及来自肌肉细胞细胞质肌球蛋白的小信号。从叠加图像中，我们得出细胞与纤维素纤维之间存在紧密相互作用的结论。我们跟踪细胞向三维结构中的迁移，表明当细胞浸入支架时，它们会在表面上方伸出丝状伪足。对致密和多孔支架的比较表明，前者的迁移深度<10 μm，而多孔类型的支架则显示细胞进一步浸入纤维素中。因此，支架结构决定了细胞整合的程度。我们得出结论，非线性显微镜可视化活的、软物质的三维组成的独特能力使其成为组织工程中的理想仪器。
