Investigating native coronary artery endothelium in situ and in cell culture by scanning force microscopy

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 °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.