Color Scanning Electron Microscopy Research Articles

Calcifi cation in the cardiovascular system is generally associated with the late stages of atherosclerosis, rheumatic fever, and aortic valve stenosis. The process that leads to calcifi cation is not completely understood, but it is widely known that calcifi c disease can impair the function of aff ected valves and arteries. The only treatment for aortic valves compromised by stenosis is surgical replacement with mechanical, bioprosthetic, or homograft valves. Homograft valves are harvested from organ donors, and usually consist of the aortic valve, part of the mitral valve, and the ascending aorta. These implants are visually inspected for structural integrity, and the absence of atheroma or calcifi c lesions. Implant candidates that do not pass this test are routinely rejected for transplant. We applied state-of-the-art characterisation methods from materials science to the homograft implants rejected for transplant, to better understand the characteristics of calcifi cation occurring in these tissues. Electron microscopy was particularly helpful in providing insight into the nature of the calcifi ed material in cardiovascular tissue, including aortic valves. This process often showed calcifi ed spherical particles (fi gure). We recorded these mineralised spheres in cardiovascular samples from patients with a diverse range of diseases, including aortic valve stenosis, rheumatic fever, and atherosclerosis. The technique that generated this colour electron micrograph is known as density-dependent colour scan ning electron microscopy, which we applied directly to histological sections from aortic valve implants rejected for transplant. We prepared the tissue by the traditional method of paraffi n embedding and microtome sectioning for 5 μm slices, then removed the paraffi n with xylene and coated the histological slide with a conductive layer of carbon, before imaging in a scanning electron microscope. The fi nal image is a combination of pictures taken in three separate modes, in a microscope equipped with diff erent electron detectors. This allows colours to be assigned according to the density of the material imaged. In the fi gure, the violet colour identifi es denser material (calcifi ed material composed of calcium phosphate), whereas structures that appear blue or green are less dense (corresponding to the extracellular matrix component of the tissue). The same multidisciplinary research approach that produced this image holds great promise for expanding our knowledge of the building blocks of calcifi c diseases in soft tissues. Lancet 2014; 384: 1294