An approach of separating the overlapped cells or nuclei based on the outer Canny edges and morphological erosion.

In biomedicine, the automatic processing of medical microscope images plays a key role in the subsequent analysis and diagnosis. Cell or nucleus segmentation is one of the most challenging tasks for microscope image processing. Due to the frequently occurred overlapping, few segmentation methods can achieve satisfactory segmentation accuracy yet. In this paper, we propose an approach to separate the overlapped cells or nuclei based on the outer Canny edges and morphological erosion. The threshold selection is first used to segment the foreground and background of cell or nucleus images. For each binary connected domain in the segmentation image, an intersection based edge selection method is proposed to choose the outer Canny edges of the overlapped cells or nuclei. The outer Canny edges are used to generate a binary cell or nucleus image that is then used to compute the cell or nucleus seeds by the proposed morphological erosion method. The nuclei of the Human U2OS cells, the mouse NIH3T3 cells and the synthetic cells are used for evaluating our proposed approach. The quantitative quantification accuracy is computed by the Dice score and 95.53% is achieved by the proposed approach. Both the quantitative and the qualitative comparisons show that the accuracy of the proposed approach is better than those of the area constrained morphological erosion (ACME) method, the iterative erosion (IE) method, the morphology and watershed (MW) method, the Generalized Laplacian of Gaussian filters (GLGF) method and ellipse fitting (EF) method in separating the cells or nuclei in three publicly available datasets.

A Deep Learning Approach for Histology-Based Nucleus Segmentation and Tumor Microenvironment Characterization.

Microscopic examination of pathology slides is essential to disease diagnosis and biomedical research. However, traditional manual examination of tissue slides is laborious and subjective. Tumor whole-slide image (WSI) scanning is becoming part of routine clinical procedures and produces massive data that capture tumor histologic details at high resolution. Furthermore, the rapid development of deep learning algorithms has significantly increased the efficiency and accuracy of pathology image analysis. In light of this progress, digital pathology is fast becoming a powerful tool to assist pathologists. Studying tumor tissue and its surrounding microenvironment provides critical insight into tumor initiation, progression, metastasis, and potential therapeutic targets. Nucleus segmentation and classification are critical to pathology image analysis, especially in characterizing and quantifying the tumor microenvironment (TME). Computational algorithms have been developed for nucleus segmentation and TME quantification within image patches. However, existing algorithms are computationally intensive and time consuming for WSI analysis. This study presents Histology-based Detection using Yolo (HD-Yolo), a new method that significantly accelerates nucleus segmentation and TME quantification. We demonstrate that HD-Yolo outperforms existing WSI analysis methods in nucleus detection, classification accuracy, and computation time. We validated the advantages of the system on 3 different tissue types: lung cancer, liver cancer, and breast cancer. For breast cancer, nucleus features by HD-Yolo were more prognostically significant than both the estrogen receptor status by immunohistochemistry and the progesterone receptor status by immunohistochemistry. The WSI analysis pipeline and a real-time nucleus segmentation viewer are available at https://github.com/impromptuRong/hd_wsi.

Transfer Learning Approach and Nucleus Segmentation with MedCLNet Colon Cancer Database.

Machine learning has been recently used especially in the medical field. In the diagnosis of serious diseases such as cancer, deep learning techniques can be used to reduce the workload of experts and to produce quick solutions. The nuclei found in the histopathology dataset are an essential parameter in disease detection. The nucleus segmentation was performed using the colorectal histology MNIST dataset for nucleus detection in this study. The graph theory, PSO, watershed, and random walker algorithms were used for the segmentation process. In addition, we present the 10-class MedCLNet visual dataset consisting of the NCT-CRC-HE-100 K dataset, LC25000 dataset, and GlaS dataset that can be used in transfer learning studies from deep learning techniques. The study proposes a transfer learning technique using the MedCLNet database. Deep neural networks pre-trained with the proposed transfer learning method were used in the classification with the colorectal histology MNIST dataset in the experimental process. DenseNet201, DenseNet169, InceptionResNetV2, InceptionV3, ResNet152V2, ResNet101V2, and Xception deep learning algorithms were used in transfer learning and the classification studies. The proposed approach was analyzed before and after transfer learning with different methods (DenseNet169 + SVM, DenseNet169 + GRU). In the performance measurement, using the colorectal histology MNIST dataset, 94.29% accuracy was obtained in the DenseNet169 model, which was initiated with random weights in the multi-classification study, and 95.00% accuracy after transfer learning was applied. In comparison with the results obtained from empirical studies, it was demonstrated that the proposed method produced satisfactory outcomes. The application is expected to provide a secondary evaluation for physicians in colon cancer detection and the segmentation.

Semantic segmentation of human cell nucleus using deep U-Net and other versions of U-Net models.

The deep learning models play an essential role in many areas, including medical image analysis. These models extract important features without human intervention. In this paper, we propose a deep convolution neural network, named as deep U-Net model, for the segmentation of the cell nucleus, a critical functional unit that determines the function and structure of the body. The nucleus contains all kinds of DNA, RNA, chromosomes, and genes governing all life activities, and its disorder may lead to different types of diseases such as cancer, heart disease, diabetes, Alzheimer's, etc. If the nucleus structure is known correctly, diseases due to nucleus disorder may be detected early. It may also reduce the drug discovery time if the shape and size of the nucleus are known. We evaluate the performance of the proposed models on the nucleus segmentation data set used by the Data Science Bowl 2018 competition hosted by Kaggle. We compare its performance with that of the U-Net, Attention U-Net, R2U-Net, Attention R2U-Net, and both versions of the U-Net++ with and without supervision, in terms of loss, dice coefficient, dice loss, intersection over union, and accuracy. Our model performs better than the existing models.

Convolutional Blur Attention Network for Cell Nuclei Segmentation.

Accurately segmented nuclei are important, not only for cancer classification, but also for predicting treatment effectiveness and other biomedical applications. However, the diversity of cell types, various external factors, and illumination conditions make nucleus segmentation a challenging task. In this work, we present a new deep learning-based method for cell nucleus segmentation. The proposed convolutional blur attention (CBA) network consists of downsampling and upsampling procedures. A blur attention module and a blur pooling operation are used to retain the feature salience and avoid noise generation in the downsampling procedure. A pyramid blur pooling (PBP) module is proposed to capture the multi-scale information in the upsampling procedure. The superiority of the proposed method has been compared with a few prior segmentation models, namely U-Net, ENet, SegNet, LinkNet, and Mask RCNN on the 2018 Data Science Bowl (DSB) challenge dataset and the multi-organ nucleus segmentation (MoNuSeg) at MICCAI 2018. The Dice similarity coefficient and some evaluation matrices, such as F1 score, recall, precision, and average Jaccard index (AJI) were used to evaluate the segmentation efficiency of these models. Overall, the proposal method in this paper has the best performance, the AJI indicator on the DSB dataset and MoNuSeg is 0.8429, 0.7985, respectively.

[Medical nucleus image segmentation network based on convolution and attention mechanism].

Although deep learning plays an important role in cell nucleus segmentation, it still faces problems such as difficulty in extracting subtle features and blurring of nucleus edges in pathological diagnosis. Aiming at the above problems, a nuclear segmentation network combined with attention mechanism is proposed. The network uses UNet network as the basic structure and the depth separable residual (DSRC) module as the feature encoding to avoid losing the boundary information of the cell nucleus. The feature decoding uses the coordinate attention (CA) to enhance the long-range distance in the feature space and highlights the key information of the nuclear position. Finally, the semantics information fusion (SIF) module integrates the feature of deep and shallow layers to improve the segmentation effect. The experiments were performed on the 2018 data science bowl (DSB2018) dataset and the triple negative breast cancer (TNBC) dataset. For the two datasets, the accuracy of the proposed method was 92.01% and 89.80%, the sensitivity was 90.09% and 91.10%, and the mean intersection over union was 89.01% and 89.12%, respectively. The experimental results show that the proposed method can effectively segment the subtle regions of the nucleus, improve the segmentation accuracy, and provide a reliable basis for clinical diagnosis.

A Deep Learning Pipeline for Nucleus Segmentation.

Deep learning is rapidly becoming the technique of choice for automated segmentation of nuclei in biological image analysis workflows. In order to evaluate the feasibility of training nuclear segmentation models on small, custom annotated image datasets that have been augmented, we have designed a computational pipeline to systematically compare different nuclear segmentation model architectures and model training strategies. Using this approach, we demonstrate that transfer learning and tuning of training parameters, such as the composition, size, and preprocessing of the training image dataset, can lead to robust nuclear segmentation models, which match, and often exceed, the performance of existing, off-the-shelf deep learning models pretrained on large image datasets. We envision a practical scenario where deep learning nuclear segmentation models trained in this way can be shared across a laboratory, facility, or institution, and continuously improved by training them on progressively larger and varied image datasets. Our work provides computational tools and a practical framework for deep learning-based biological image segmentation using small annotated image datasets. Published [2020]. This article is a U.S. Government work and is in the public domain in the USA.

Object-Oriented Segmentation of Cell Nuclei in Fluorescence Microscopy Images.

Cell nucleus segmentation remains an open and challenging problem especially to segment nuclei in cell clumps. Splitting a cell clump would be straightforward if the gradients of boundary pixels in-between the nuclei were always higher than the others. However, imperfections may exist: inhomogeneities of pixel intensities in a nucleus may cause to define spurious boundaries whereas insufficient pixel intensity differences at the border of overlapping nuclei may cause to miss some true boundary pixels. In contrast, these imperfections are typically observed at the pixel-level, causing local changes in pixel values without changing the semantics on a large scale. In response to these issues, this article introduces a new nucleus segmentation method that relies on using gradient information not at the pixel level but at the object level. To this end, it proposes to decompose an image into smaller homogeneous subregions, define edge-objects at four different orientations to encode the gradient information at the object level, and devise a merging algorithm, in which the edge-objects vote for subregion pairs along their orientations and the pairs are iteratively merged if they get sufficient votes from multiple orientations. Our experiments on fluorescence microscopy images reveal that this high-level representation and the design of a merging algorithm using edge-objects (gradients at the object level) improve the segmentation results.

Iterative h-minima-based marker-controlled watershed for cell nucleus segmentation.

Automated microscopy imaging systems facilitate high-throughput screening in molecular cellular biology research. The first step of these systems is cell nucleus segmentation, which has a great impact on the success of the overall system. The marker-controlled watershed is a technique commonly used by the previous studies for nucleus segmentation. These studies define their markers finding regional minima on the intensity/gradient and/or distance transform maps. They typically use the h-minima transform beforehand to suppress noise on these maps. The selection of the h value is critical; unnecessarily small values do not sufficiently suppress the noise, resulting in false and oversegmented markers, and unnecessarily large ones suppress too many pixels, causing missing and undersegmented markers. Because cell nuclei show different characteristics within an image, the same h value may not work to define correct markers for all the nuclei. To address this issue, in this work, we propose a new watershed algorithm that iteratively identifies its markers, considering a set of different h values. In each iteration, the proposed algorithm defines a set of candidates using a particular h value and selects the markers from those candidates provided that they fulfill the size requirement. Working with widefield fluorescence microscopy images, our experiments reveal that the use of multiple h values in our iterative algorithm leads to better segmentation results, compared to its counterparts. © 2016 International Society for Advancement of Cytometry.

ClusterSeg: A crowd cluster pinpointed nucleus segmentation framework with cross-modality datasets.

The detection and segmentation of individual cells or nuclei is often involved in image analysis across a variety of biology and biomedical applications as an indispensable prerequisite. However, the ubiquitous presence of crowd clusters with morphological variations often hinders successful instance segmentation. In this paper, nuclei cluster focused annotation strategies and frameworks are proposed to overcome this challenging practical problem. Specifically, we design a nucleus segmentation framework, namely ClusterSeg, to tackle nuclei clusters, which consists of a convolutional-transformer hybrid encoder and a 2.5-path decoder for precise predictions of nuclei instance mask, contours, and clustered-edges. Additionally, an annotation-efficient clustered-edge pointed strategy pinpoints the salient and error-prone boundaries, where a partially-supervised PS-ClusterSeg is presented using ClusterSeg as the segmentation backbone. The framework is evaluated with four privately curated image sets and two public sets with characteristic severely clustered nuclei across a variety range of image modalities, e.g., microscope, cytopathology, and histopathology images. The proposed ClusterSeg and PS-ClusterSeg are modality-independent and generalizable, and superior to current state-of-the-art approaches in multiple metrics empirically. Our collected data, the elaborate annotations to both public and private set, as well the source code, are released publicly at https://github.com/lu-yizhou/ClusterSeg.

NuKit: A deep learning platform for fast nucleus segmentation of histopathological images.

Nucleus segmentation represents the initial step for histopathological image analysis pipelines, and it remains a challenge in many quantitative analysis methods in terms of accuracy and speed. Recently, deep learning nucleus segmentation methods have demonstrated to outperform previous intensity- or pattern-based methods. However, the heavy computation of deep learning provides impression of lagging response in real time and hampered the adoptability of these models in routine research. We developed and implemented NuKit a deep learning platform, which accelerates nucleus segmentation and provides prompt results to the users. NuKit platform consists of two deep learning models coupled with an interactive graphical user interface (GUI) to provide fast and automatic nucleus segmentation "on the fly". Both deep learning models provide complementary tasks in nucleus segmentation. The whole image segmentation model performs whole image nucleus whereas the click segmentation model supplements the nucleus segmentation with user-driven input to edits the segmented nuclei. We trained the NuKit whole image segmentation model on a large public training data set and tested its performance in seven independent public image data sets. The whole image segmentation model achieves average [Formula: see text] and [Formula: see text]. The outputs could be exported into different file formats, as well as provides seamless integration with other image analysis tools such as QuPath. NuKit can be executed on Windows, Mac, and Linux using personal computers.

BAWGNet: Boundary aware wavelet guided network for the nuclei segmentation in histopathology images.

Precise cell nucleus segmentation is very critical in many biologically related analyses and disease diagnoses. However, the variability in nuclei structure, color, and modalities of histopathology images make the automatic computer-aided nuclei segmentation task very difficult. Traditional encoder-decoder based deep learning schemes mainly utilize the spatial domain information that may limit the performance of recognizing small nuclei regions in subsequent downsampling operations. In this paper, a boundary aware wavelet guided network (BAWGNet) is proposed by incorporating a boundary aware unit along with an attention mechanism based on a wavelet domain guidance in each stage of the encoder-decoder output. Here the high-frequency 2 Dimensional discrete wavelet transform (2D-DWT) coefficients are utilized in the attention mechanism to guide the spatial information obtained from the encoder-decoder output stages to leverage the nuclei segmentation task. On the other hand, the boundary aware unit (BAU) captures the nuclei's boundary information, ensuring accurate prediction of the nuclei pixels in the edge region. Furthermore, the preprocessing steps used in our methodology confirm the data's uniformity by converting it to similar color statistics. Extensive experimentations conducted on three benchmark histopathology datasets (DSB, MoNuSeg and TNBC) exhibit the outstanding segmentation performance of the proposed method (with dice scores 90.82%, 85.74%, and 78.57%, respectively). Implementation of the proposed architecture is available at https://github.com/tamjidimtiaz/BAWGNet.

Predicting Crohn's disease severity in the colon using mixed cell nucleus density from pseudo labels.

Crohn's disease (CD) is a debilitating inflammatory bowel disease with no known cure. Computational analysis of hematoxylin and eosin (H&E) stained colon biopsy whole slide images (WSIs) from CD patients provides the opportunity to discover unknown and complex relationships between tissue cellular features and disease severity. While there have been works using cell nuclei-derived features for predicting slide-level traits, this has not been performed on CD H&E WSIs for classifying normal tissue from CD patients vs active CD and assessing slide label-predictive performance while using both separate and combined information from pseudo-segmentation labels of nuclei from neutrophils, eosinophils, epithelial cells, lymphocytes, plasma cells, and connective cells. We used 413 WSIs of CD patient biopsies and calculated normalized histograms of nucleus density for the six cell classes for each WSI. We used a support vector machine to classify the truncated singular value decomposition representations of the normalized histograms as normal or active CD with four-fold cross-validation in rounds where nucleus types were first compared individually, the best was selected, and further types were added each round. We found that neutrophils were the most predictive individual nucleus type, with an AUC of 0.92 ± 0.0003 on the withheld test set. Adding information improved cross-validation performance for the first two rounds and on the withheld test set for the first three rounds, though performance metrics did not increase substantially beyond when neutrophils were used alone.

An Innovative Solution Based on TSCA-ViT for Osteosarcoma Diagnosis in Resource-Limited Settings.

Identifying and managing osteosarcoma pose significant challenges, especially in resource-constrained developing nations. Advanced diagnostic methods involve isolating the nucleus from cancer cells for comprehensive analysis. However, two main challenges persist: mitigating image noise during the capture and transmission of cellular sections, and providing an efficient, accurate, and cost-effective solution for cell nucleus segmentation. To tackle these issues, we introduce the Twin-Self and Cross-Attention Vision Transformer (TSCA-ViT). This pioneering AI-based system employs a directed filtering algorithm for noise reduction and features an innovative transformer architecture with a twin attention mechanism for effective segmentation. The model also incorporates cross-attention-enabled skip connections to augment spatial information. We evaluated our method on a dataset of 1000 osteosarcoma pathology slide images from the Second People's Hospital of Huaihua, achieving a remarkable average precision of 97.7%. This performance surpasses traditional methodologies. Furthermore, TSCA-ViT offers enhanced computational efficiency owing to its fewer parameters, which results in reduced time and equipment costs. These findings underscore the superior efficacy and efficiency of TSCA-ViT, offering a promising approach for addressing the ongoing challenges in osteosarcoma diagnosis and treatment, particularly in settings with limited resources.

A survey on recent trends in deep learning for nucleus segmentation from histopathology images.

Nucleus segmentation is an imperative step in the qualitative study of imaging datasets, considered as an intricate task in histopathology image analysis. Segmenting a nucleus is an important part of diagnosing, staging, and grading cancer, but overlapping regions make it hard to separate and tell apart independent nuclei. Deep Learning is swiftly paving its way in the arena of nucleus segmentation, attracting quite a few researchers with its numerous published research articles indicating its efficacy in the field. This paper presents a systematic survey on nucleus segmentation using deep learning in the last five years (2017-2021), highlighting various segmentation models (U-Net, SCPP-Net, Sharp U-Net, and LiverNet) and exploring their similarities, strengths, datasets utilized, and unfolding research areas.

NuCLS: A scalable crowdsourcing approach and dataset for nucleus classification and segmentation in breast cancer.

BACKGROUND: Deep learning enables accurate high-resolution mapping of cells and tissue structures that can serve as the foundation of interpretable machine-learning models for computational pathology. However, generating adequate labels for these structures is a critical barrier, given the time and effort required from pathologists. RESULTS: This article describes a novel collaborative framework for engaging crowds of medical students and pathologists to produce quality labels for cell nuclei. We used this approach to produce the NuCLS dataset, containing >220,000 annotations of cell nuclei in breast cancers. This builds on prior work labeling tissue regions to produce an integrated tissue region- and cell-level annotation dataset for training that is the largest such resource for multi-scale analysis of breast cancer histology. This article presents data and analysis results for single and multi-rater annotations from both non-experts and pathologists. We present a novel workflow that uses algorithmic suggestions to collect accurate segmentation data without the need for laborious manual tracing of nuclei. Our results indicate that even noisy algorithmic suggestions do not adversely affect pathologist accuracy and can help non-experts improve annotation quality. We also present a new approach for inferring truth from multiple raters and show that non-experts can produce accurate annotations for visually distinctive classes. CONCLUSIONS: This study is the most extensive systematic exploration of the large-scale use of wisdom-of-the-crowd approaches to generate data for computational pathology applications.

An Integrative Segmentation Framework for Cell Nucleus of Fluorescence Microscopy.

Nucleus segmentation of fluorescence microscopy is a critical step in quantifying measurements in cell biology. Automatic and accurate nucleus segmentation has powerful applications in analyzing intrinsic characterization in nucleus morphology. However, existing methods have limited capacity to perform accurate segmentation in challenging samples, such as noisy images and clumped nuclei. In this paper, inspired by the idea of cascaded U-Net (or W-Net) and its remarkable performance improvement in medical image segmentation, we proposed a novel framework called Attention-enhanced Simplified W-Net (ASW-Net), in which a cascade-like structure with between-net connections was used. Results showed that this lightweight model could reach remarkable segmentation performance in the BBBC039 testing set (aggregated Jaccard index, 0.90). In addition, our proposed framework performed better than the state-of-the-art methods in terms of segmentation performance. Moreover, we further explored the effectiveness of our designed network by visualizing the deep features from the network. Notably, our proposed framework is open source.

Nucleus segmentation: towards automated solutions.

Single nucleus segmentation is a frequent challenge of microscopy image processing, since it is the first step of many quantitative data analysis pipelines. The quality of tracking single cells, extracting features or classifying cellular phenotypes strongly depends on segmentation accuracy. Worldwide competitions have been held, aiming to improve segmentation, and recent years have definitely brought significant improvements: large annotated datasets are now freely available, several 2D segmentation strategies have been extended to 3D, and deep learning approaches have increased accuracy. However, even today, no generally accepted solution and benchmarking platform exist. We review the most recent single-cell segmentation tools, and provide an interactive method browser to select the most appropriate solution.

Combining simple interactivity and machine learning: a separable deep learning approach to subthalamic nucleus localization and segmentation in MRI for deep brain stimulation surgical planning.

Purpose: Deep brain stimulation (DBS) is an interventional treatment for some neurological and neurodegenerative diseases. For example, in Parkinson's disease, DBS electrodes are positioned at particular locations within the basal ganglia to alleviate the patient's motor symptoms. These interventions depend greatly on a preoperative planning stage in which potential targets and electrode trajectories are identified in a preoperative MRI. Due to the small size and low contrast of targets such as the subthalamic nucleus (STN), their segmentation is a difficult task. Machine learning provides a potential avenue for development, but it has difficulty in segmenting such small structures in volumetric images due to additional problems such as segmentation class imbalance. Approach: We present a two-stage separable learning workflow for STN segmentation consisting of a localization step that detects the STN and crops the image to a small region and a segmentation step that delineates the structure within that region. The goal of this decoupling is to improve accuracy and efficiency and to provide an intermediate representation that can be easily corrected by a clinical user. This correction capability was then studied through a human-computer interaction experiment with seven novice participants and one expert neurosurgeon. Results: Our two-step segmentation significantly outperforms the comparative registration-based method currently used in clinic and approaches the fundamental limit on variability due to the image resolution. In addition, the human-computer interaction experiment shows that the additional interaction mechanism allowed by separating STN segmentation into two steps significantly improves the users' ability to correct errors and further improves performance. Conclusions: Our method shows that separable learning not only is feasible for fully automatic STN segmentation but also leads to improved interactivity that can ease its translation into clinical use.

Improved Quantification of Cell Density in the Arterial Wall-A Novel Nucleus Splitting Approach Applied to 3D Two-Photon Laser-Scanning Microscopy.

Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a "nucleus splitting" procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR Apoe -/- and control Apoe -/- mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed -26.6 ± 9.90% (p < 0.001) and -1.44 ± 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.