Postmortem high-resolution 3-dimensional imaging of the primate brain: blockface imaging of perfusion stained tissue

Modern anatomy of the brain is primarily concerned with visualizing contrast between tissue elements and with 3-dimensional (3-D) reconstruction. The first objective relies on specific neuroanatomical and imaging protocols, the latter on proper alignment of serial cross-sectional anatomy. We present a method that meets these goals at a resolution that allows for the structural delineation in the primate central nervous system. Methylene blue was administered by perfusion to the intact brain of three rhesus macaques thereby staining the neuropil in the cortex and gray nuclei of the brain in situ. Subsequent dissection and serial sectioning revealed excellent contrast in the tissue and showed clear boundaries between anatomical structures. We captured sequential images of the block surface of the brain before each serial section was cut from the specimen by means of a digital camera mounted directly over the microtome stage and the brain. We developed a suite of algorithms to automate the alignment, segmentation, normalization, and intensity correction for the series of blockface images. These digital images were the basis for 3-D reconstruction of the brains.