Application of 3D optical sectioning microscopy to the study of microvascular biology
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The microvasculature is characterized by a highly-organized three-dimensional (3D) array of interconnecting blood vessels. Studies of the microcirculation using conventional intravital microscopy techniques provide two-dimensional (2D) views of its architecture. These 2D views lack information about the axial dimension and therefore underrepresent the spatial relationships that exist between vessels and their parenchymal environment and between different classes of vessels. 3D information would also be an invaluable adjunct to studies of events occurring at the level of vascular wall. Here a lack of differentiation in the axial dimension can interfere with elucidation of mechanical events and complex cell-to-cell or cell-matrix relationships. With the advent of optical sectioning techniques it is now possible to obtain a series of images with each representing a different focal depth. This is achieved by using a digital imaging microscope equipped with computer control of the focus and the camera readout for image acquisition. High resolution images are obtained with a cooled-CCD camera, digitized and stored for computer processing and 3D reconstruction. A graphics workstation is used to manipulate the 3D data set to display various views of interest and for feature extraction and analysis. With computerized control of the microscope stage the optical sectioning can be combined with automated orthogonal sampling. This permits collection of a series of spatially-related images from a single orthogonal image plane. These images can then be combined into a single montage view of a large area. In summary, these imaging techniques promise to be powerful new tools for the study of the microcirculation. (Supp HL33324 & AHA-Est. Invest. Awd. to G.A.M.)
