Reichlin Tobias, Wild Andreas, Dürrenberger Markus, Daniels A U, Aebi Ueli, Hunziker Patrick R, Stolz Martin
M.E. Müller Institute for Structural Biology, Biozentrum University of Basel, Switzerland.
J Struct Biol. 2005 Oct;152(1):52-63. doi: 10.1016/j.jsb.2005.07.009.
The purpose of our studies is to better understand the morphology and functioning of the arteries and their changes in pathogenesis. The most frequently used imaging techniques are intravascular ultrasound, magnetic resonance imaging, and optical coherence tomography. These methods do not image cell-level structural details and only provide biomechanical properties indirectly. We present a new protocol for imaging the endothelial surface and measuring elastic properties of vascular tissue by scanning force microscopy. Full-thickness sections of native pig coronary arteries were prepared. In addition, cultured human umbilical vein endothelial cells were studied as an in vitro model system and for comparison. We encountered a variety of difficulties mostly due to the softness of vascular tissue which required significant adaptations of standard equipment: (i) a new specimen holder designed to stably immobilize the coronary arteries; (ii) a phase-contrast microscope incorporated for assessing the status of the cultured endothelial cells and positioning the scanning force microscope (SFM) tip at a site of interest; and (iii) a continuous exchange of the culture medium at 37 degrees C to assure viability of the cells in the SFM over extended times. We were thus able to investigate both fresh arterial tissue and living endothelial cells in a near-physiological environment. We present initial SFM images of vascular tissue at a spatial resolution similar to scanning electron microscopy, but which also provide a closer view of the bona fide structure of native tissue. Novel morphological features such as distinct granular particles were observed. Moreover, we report initial measurements of vascular tissue surface stiffness, obtained by indentation-type SFM.
我们研究的目的是更好地了解动脉的形态和功能以及它们在发病机制中的变化。最常用的成像技术是血管内超声、磁共振成像和光学相干断层扫描。这些方法无法成像细胞水平的结构细节，只能间接提供生物力学特性。我们提出了一种通过扫描力显微镜对内皮表面进行成像并测量血管组织弹性特性的新方案。制备了天然猪冠状动脉的全层切片。此外，研究了培养的人脐静脉内皮细胞作为体外模型系统并用于比较。我们遇到了各种困难，主要是由于血管组织的柔软性，这需要对标准设备进行重大调整：（i）设计了一种新的标本架，用于稳定固定冠状动脉；（ii）并入一台相差显微镜，用于评估培养的内皮细胞的状态并将扫描力显微镜（SFM）尖端定位在感兴趣的部位；（iii）在37摄氏度下持续更换培养基，以确保SFM中的细胞在较长时间内保持活力。因此，我们能够在近乎生理的环境中研究新鲜动脉组织和活的内皮细胞。我们展示了血管组织的初始SFM图像，其空间分辨率与扫描电子显微镜相似，但也能更近距离地观察天然组织的真实结构。观察到了一些新的形态特征，如明显的颗粒状颗粒。此外，我们报告了通过压痕型SFM获得的血管组织表面硬度的初始测量结果。
