The centroids of putative nuclear profiles estimated from 2D images were then grouped across z planes in an image stack to identify the positions of nuclei in x– y– z. DAPI channel images were automatically thresholded, and contiguous regions that included multiple clumped nuclear profiles were split using k-means clustering of image pixels for a set of candidate k values determined based on the clump’s area the most likely candidate segmentation was selected based on criteria that capture expected nuclear profile shape and size. A series of overlapping confocal image stacks were acquired, each spanning from the pial surface to the white matter. Its utility was demonstrated by quantifying neuronal density in macaque cortical areas V1 and V2. ![]() A new framework for measuring densities of immunolabeled neurons across cortical layers was implemented that combines a confocal microscopy sampling strategy with automated analysis of 3D image stacks.
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