Segmentor: a tool for manual refinement of 3D microscopy annotations.

BACKGROUND: Recent advances in tissue clearing techniques, combined with high-speed image acquisition through light sheet microscopy, enable rapid three-dimensional (3D) imaging of biological specimens, such as whole mouse brains, in a matter of hours. Quantitative analysis of such 3D images can help us understand how changes in brain structure lead to differences in behavior or cognition, but distinguishing densely packed features of interest, such as nuclei, from background can be challenging. Recent deep learning-based nuclear segmentation algorithms show great promise for automated segmentation, but require large numbers of accurate manually labeled nuclei as training data. RESULTS: We present Segmentor, an open-source tool for reliable, efficient, and user-friendly manual annotation and refinement of objects (e.g., nuclei) within 3D light sheet microscopy images. Segmentor employs a hybrid 2D-3D approach for visualizing and segmenting objects and contains features for automatic region splitting, designed specifically for streamlining the process of 3D segmentation of nuclei. We show that editing simultaneously in 2D and 3D using Segmentor significantly decreases time spent on manual annotations without affecting accuracy as compared to editing the same set of images with only 2D capabilities. CONCLUSIONS: Segmentor is a tool for increased efficiency of manual annotation and refinement of 3D objects that can be used to train deep learning segmentation algorithms, and is available at https://www.nucleininja.org/ and https://github.com/RENCI/Segmentor .

Influence of Brachial Plexus Birth Injury Location on Glenohumeral Joint Morphology.

PURPOSE: Patient presentation after brachial plexus birth injury (BPBI) is influenced by nerve injury location; more contracture and bone deformity occur at the shoulder in postganglionic injuries. Although bone deformity after postganglionic injury is well-characterized, the extent of glenohumeral deformity after preganglionic BPBI is unclear. METHODS: Twenty Sprague-Dawley rat pups received preganglionic or postganglionic neurectomy on a single forelimb at postnatal days 3 to 4. Glenohumeral joints on affected and unaffected sides were analyzed using micro-computed tomography scans after death at 8 weeks after birth. Glenoid version, glenoid inclination, glenoid and humeral head radius of curvature, and humeral head thickness and width were measured bilaterally. RESULTS: The glenoid was significantly more declined in affected compared with unaffected shoulders after postganglionic (-17.7° ± 16.9°) but not preganglionic injury. Compared with the preganglionic group, the affected shoulder in the postganglionic group exhibited significantly greater declination and increased glenoid radius of curvature. In contrast, the humeral head was only affected after preganglionic but not postganglionic injury, with a significantly smaller humeral head radius of curvature (-0.2 ± 0.2 mm), thickness (-0.2 ± 0.3 mm), and width (-0.3 ± 0.4 mm) on the affected side compared with the unaffected side; changes in these metrics were significantly associated with each other. CONCLUSIONS: These findings suggest that glenoid deformities occur after postganglionic BPBI but not after preganglionic BPBI, whereas the humeral head is smaller after preganglionic injury, possibly suggesting an overall decreased biological growth rate in this group. CLINICAL RELEVANCE: This study expands understanding of the altered glenoid and humeral head morphologies after preganglionic BPBI and its comparisons with morphologies after postganglionic BPBI.

Preganglionic and Postganglionic Brachial Plexus Birth Injury Effects on Shoulder Muscle Growth.

PURPOSE: Brachial plexus birth injury can differ in presentation, depending on whether the nerve ruptures distal to, or avulses proximal to, the dorsal root ganglion. More substantial contracture and bone deformity at the shoulder is typical in postganglionic injuries. However, changes to the underlying muscle structure that drive these differences in presentation are unclear. METHODS: Seventeen Sprague-Dawley rats received preganglionic or postganglionic neurectomy on a single limb on postnatal days 3 and 4. Muscles crossing the shoulder were retrieved once the rats were sacrificed at 8 weeks after birth. External rotation range of motion, muscle mass, muscle length, muscle sarcomere length, and calculated optimal muscle length were measured bilaterally. RESULTS: Average shoulder range of motion in the postganglionic group was 61.8% and 56.2% more restricted at 4 and 8 weeks, respectively, compared with that in the preganglionic group, but affected muscles after preganglionic injury were altered more severely (compared with the unaffected limb) than after postganglionic injury. Optimal muscle length in preganglionic injury was shorter in the affected limb (compared with the unaffected limb: -18.2% ± 9.2%) and to a greater extent than in postganglionic injury (-5.1% ± 6.2%). Muscle mass in preganglionic injury was lower in the affected limb (relative to the unaffected limb: -57.2% ± 24.1%) and to a greater extent than in postganglionic injury (-28.1% ± 17.7%). CONCLUSIONS: The findings suggest that the presence of contracture does not derive from restricted longitudinal muscle growth alone, but also depends on the extent of muscle mass loss occurring simultaneously after the injury. CLINICAL RELEVANCE: This study expands our understanding of differences in muscle architecture and the role of muscle structure in contracture formation for preganglionic and postganglionic brachial plexus birth injury.

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei. Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases. We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.