Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.

Level set segmentation of breast masses in contrast-enhanced dedicated breast CT and evaluation of stopping criteria.

Dedicated breast CT (bCT) produces high-resolution 3D tomographic images of the breast, fully resolving fibroglandular tissue structures within the breast and allowing for breast lesion detection and assessment in 3D. In order to enable quantitative analysis, such as volumetrics, automated lesion segmentation on bCT is highly desirable. In addition, accurate output from CAD (computer-aided detection/diagnosis) methods depends on sufficient segmentation of lesions. Thus, in this study, we present a 3D lesion segmentation method for breast masses in contrast-enhanced bCT images. The segmentation algorithm follows a two-step approach. First, 3D radial-gradient index segmentation is used to obtain a crude initial contour, which is then refined by a 3D level set-based active contour algorithm. The data set included contrast-enhanced bCT images from 33 patients containing 38 masses (25 malignant, 13 benign). The mass centers served as input to the algorithm. In this study, three criteria for stopping the contour evolution were compared, based on (1) the change of region volume, (2) the average intensity in the segmented region increase at each iteration, and (3) the rate of change of the average intensity inside and outside the segmented region. Lesion segmentation was evaluated by computing the overlap ratio between computer segmentations and manually drawn lesion outlines. For each lesion, the overlap ratio was averaged across coronal, sagittal, and axial planes. The average overlap ratios for the three stopping criteria ranged from 0.66 to 0.68 (dice coefficient of 0.80 to 0.81), indicating that the proposed segmentation procedure is promising for use in quantitative dedicated bCT analyses.

Regional and cell-type-specific afferent and efferent projections of the mouse claustrum.

The claustrum (CLA) is a conspicuous subcortical structure interconnected with cortical and subcortical regions. Its regional anatomy and cell-type-specific connections in the mouse remain not fully determined. Using multimodal reference datasets, we confirmed the delineation of the mouse CLA as a single group of neurons embedded in the agranular insular cortex. We quantitatively investigated brain-wide inputs and outputs of CLA using bulk anterograde and retrograde viral tracing data and single neuron tracing data. We found that the prefrontal module has more cell types projecting to the CLA than other cortical modules, with layer 5 IT neurons predominating. We found nine morphological types of CLA principal neurons that topographically innervate functionally linked cortical targets, preferentially the midline cortical areas, secondary motor area, and entorhinal area. Together, this study provides a detailed wiring diagram of the cell-type-specific connections of the mouse CLA, laying a foundation for studying its functions at the cellular level.

Connecting single-cell transcriptomes to projectomes in mouse visual cortex.

The mammalian brain is composed of diverse neuron types that play different functional roles. Recent single-cell RNA sequencing approaches have led to a whole brain taxonomy of transcriptomically-defined cell types, yet cell type definitions that include multiple cellular properties can offer additional insights into a neuron's role in brain circuits. While the Patch-seq method can investigate how transcriptomic properties relate to the local morphological and electrophysiological properties of cell types, linking transcriptomic identities to long-range projections is a major unresolved challenge. To address this, we collected coordinated Patch-seq and whole brain morphology data sets of excitatory neurons in mouse visual cortex. From the Patch-seq data, we defined 16 integrated morpho-electric-transcriptomic (MET)-types; in parallel, we reconstructed the complete morphologies of 300 neurons. We unified the two data sets with a multi-step classifier, to integrate cell type assignments and interrogate cross-modality relationships. We find that transcriptomic variations within and across MET-types correspond with morphological and electrophysiological phenotypes. In addition, this variation, along with the anatomical location of the cell, can be used to predict the projection targets of individual neurons. We also shed new light on infragranular cell types and circuits, including cell-type-specific, interhemispheric projections. With this approach, we establish a comprehensive, integrated taxonomy of excitatory neuron types in mouse visual cortex and create a system for integrated, high-dimensional cell type classification that can be extended to the whole brain and potentially across species.

DeepNeuron: an open deep learning toolbox for neuron tracing.

Reconstructing three-dimensional (3D) morphology of neurons is essential for understanding brain structures and functions. Over the past decades, a number of neuron tracing tools including manual, semiautomatic, and fully automatic approaches have been developed to extract and analyze 3D neuronal structures. Nevertheless, most of them were developed based on coding certain rules to extract and connect structural components of a neuron, showing limited performance on complicated neuron morphology. Recently, deep learning outperforms many other machine learning methods in a wide range of image analysis and computer vision tasks. Here we developed a new Open Source toolbox, DeepNeuron, which uses deep learning networks to learn features and rules from data and trace neuron morphology in light microscopy images. DeepNeuron provides a family of modules to solve basic yet challenging problems in neuron tracing. These problems include but not limited to: (1) detecting neuron signal under different image conditions, (2) connecting neuronal signals into tree(s), (3) pruning and refining tree morphology, (4) quantifying the quality of morphology, and (5) classifying dendrites and axons in real time. We have tested DeepNeuron using light microscopy images including bright-field and confocal images of human and mouse brain, on which DeepNeuron demonstrates robustness and accuracy in neuron tracing.

Impact of lesion segmentation metrics on computer-aided diagnosis/detection in breast computed tomography.

Evaluation of segmentation algorithms usually involves comparisons of segmentations to gold-standard delineations without regard to the ultimate medical decision-making task. We compare two segmentation evaluations methods-a Dice similarity coefficient (DSC) evaluation and a diagnostic classification task-based evaluation method using lesions from breast computed tomography. In our investigation, we use results from two previously developed lesion-segmentation algorithms [a global active contour model (GAC) and a global with local aspects active contour model]. Although similar DSC values were obtained (0.80 versus 0.77), we show that the global + local active contour (GLAC) model, as compared with the GAC model, is able to yield significantly improved classification performance in terms of area under the receivers operating characteristic (ROC) curve in the task of distinguishing malignant from benign lesions. [Area under the [Formula: see text] compared to 0.63, [Formula: see text]]. This is mainly because the GLAC model yields better detailed information required in the calculation of morphological features. Based on our findings, we conclude that the DSC metric alone is not sufficient for evaluating segmentation lesions in computer-aided diagnosis tasks.

Segmentation of breast masses on dedicated breast computed tomography and three-dimensional breast ultrasound images.

We present and evaluate a method for the three-dimensional (3-D) segmentation of breast masses on dedicated breast computed tomography (bCT) and automated 3-D breast ultrasound images. The segmentation method, refined from our previous segmentation method for masses on contrast-enhanced bCT, includes two steps: (1) initial contour estimation and (2) active contour-based segmentation to further evolve and refine the initial contour by adding a local energy term to the level-set equation. Segmentation performance was assessed in terms of Dice coefficients (DICE) for 129 lesions on noncontrast bCT, 38 lesions on contrast-enhanced bCT, and 98 lesions on 3-D breast ultrasound (US) images. For bCT, DICE values of 0.82 and 0.80 were obtained on contrast-enhanced and noncontrast images, respectively. The improvement in segmentation performance with respect to that of our previous method was statistically significant ( p = 0.002 ). Moreover, segmentation appeared robust with respect to the presence of glandular tissue. For 3-D breast US, the DICE value was 0.71. Hence, our method obtained promising results for both 3-D imaging modalities, laying a solid foundation for further quantitative image analysis and potential future expansion to other 3-D imaging modalities.

DBCAT: database of CpG islands and analytical tools for identifying comprehensive methylation profiles in cancer cells.

DBCAT (database of CpG islands and analytical tools, http://dbcat.cgm.ntu.edu.tw/ ), developed to characterize comprehensive DNA methylation profiles in human cancers, is a web-based application and methylation database containing several convenient tools for investigating epigenetic regulation in human diseases. To our knowledge, DBCAT is the first online methylation analytical tool, and is composed of three parts: a CpG island finder, a genome query browser, and a tool for analyzing methylation microarray data. The analytical tools can quickly identify genes with methylated regions from microarray data, compare the methylation status changes between different arrays, and provide functional analysis in addition to colocalizing transcription factor binding sites.