The purpose of this study was to evaluate the use of laser scanning confocal fluorescence microscopy for the assessment of cardiovascular device-related pathology. This emerging technology allows for the simultaneous multicolor imaging of paraffin or frozen sections stained with fluorochrome-labeled antibodies and/or specific fluorescent reagents. This imaging can be combined with other modes of microscopy, such as Nomarski differential interference contrast (DIC), phase contrast or incident polarized light microscopy. In addition, Z-scans (optical cross sections at multiple levels within the tissue section) can be obtained and used for three-dimensional reconstructions of the topographic distribution of the fluorescent labeling. Through the appropriate selection of primary and secondary antibodies or specific stains (e.g., for nuclear DNA) and laser excitation and fluorescence emission wavelengths, confocal microscopy makes it possible to visualize up to four fluorescent markers or stains, with minimal fluorescent quenching (fading or photobleaching). We routinely employ a confocal microscope to obtain images of histologic sections stained using secondary antibodies labeled with fluorescein isothiocyanate (FITC; green fluorescence) and Texas Red (red fluorescence) and nuclear DNA stains with 2,6-diamidino-4-phenylindole (DAPI; blue fluorescence). These methods provide unique information concerning the coloralization of intra- or extracellular components in normal and pathological conditions. We have used this technique to study the changes that develop in allograft heart valves after implantation in sheep. We are in the processing of developing other applications of this technique for the study of: 1) the biological and pathological effects of cardiovascular prosthetic devices being developed as viable tissue- engineered prostheses (e.g., blood vessels and heart valves), and 2) the effects of biomechanical factors on tissue remodeling in valved conduits or within vascular walls in response to angioplasty or the placement of vascular stents or stented-grafts. - confocal microscopy; prosthetic heart valves; factor VIII; matrix metalloproteinases; immunohistochemistry
