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1.  Serial section registration of axonal confocal microscopy datasets for long-range neural circuit reconstruction 
Journal of neuroscience methods  2012;207(2):200-210.
In the context of long-range digital neural circuit reconstruction, this paper investigates an approach for registering axons across histological serial sections. Tracing distinctly labeled axons over large distances allows neuroscientists to study very explicit relationships between the brain’s complex interconnects and, for example, diseases or aberrant development. Large scale histological analysis requires, however, that the tissue be cut into sections. In immunohistochemical studies thin sections are easily distorted due to the cutting, preparation, and slide mounting processes. In this work we target the registration of thin serial sections containing axons. Sections are first traced to extract axon centerlines, and these traces are used to define registration landmarks where they intersect section boundaries. The trace data also provides distinguishing information regarding an axon’s size and orientation within a section. We propose the use of these features when pairing axons across sections in addition to utilizing the spatial relationships among the landmarks. The global rotation and translation of an unregistered section are accounted for using a random sample consensus (RANSAC) based technique. An iterative nonrigid refinement process using B-spline warping is then used to reconnect axons and produce the sought after connectivity information.
PMCID: PMC4981587  PMID: 22465678
Serial section registration; Long-range neural circuits
2.  Pepsin Pretreatment Allows Collagen IV Immunostaining of Blood Vessels in Adult Mouse Brain 
Journal of neuroscience methods  2007;163(1):76-82.
While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.
PMCID: PMC1931483  PMID: 17403541
adult; antigen retrieval; blood vessels; brain; collagen IV; immunohistochemistry; mouse; pepsin
3.  Three-Dimensional Neuron Tracing by Voxel Scooping 
Journal of neuroscience methods  2009;184(1):169-175.
Tracing the centerline of the dendritic arbor of neurons is a powerful technique for analyzing neuronal morphology. In the various neuron tracing algorithms in use nowadays, the competing goals of computational efficiency and robustness are generally traded off against each other. We present a novel method for tracing the centerline of a neuron from confocal image stacks, which provides an optimal balance between these objectives. Using only local information, thin cross-sectional layers of voxels (‘scoops’) are iteratively carved out of the structure, and clustered based on connectivity. Each cluster contributes a node along the centerline, which is created by connecting successive nodes until all object voxels are exhausted. While data segmentation is independent of this algorithm, we illustrate the use of the ISODATA method to achieve dynamic (local) segmentation. Diameter estimation at each node is calculated using the Rayburst Sampling algorithm, and spurious end nodes caused by surface irregularities are then removed. On standard computing hardware the algorithm can process hundreds of thousands of voxels per second, easily handling the multi-gigabyte datasets resulting from high-resolution confocal microscopy imaging of neurons. This method provides an accurate and efficient means for centerline extraction that is suitable for interactive neuron tracing applications.
PMCID: PMC2753723  PMID: 19632273
Centerline Extraction; Neuron Tracing; Image Analysis; Medial Axis

Results 1-3 (3)