Cell boundary information is crucial for analyzing cell behaviors from time-lapse microscopy videos. Existing supervised cell segmentation tools, such as ImageJ, require tuning various parameters and rely on restrictive assumptions about the shape of the objects. While recent supervised segmentation tools based on convolutional neural networks enhance accuracy, they depend on high-quality labelled images, making them unsuitable for segmenting new types of objects not in the database. We developed a novel unsupervised cell segmentation algorithm based on fast Gaussian processes for noisy microscopy images without the need for parameter tuning or restrictive assumptions about the shape of the object. We derived robust thresholding criteria adaptive for heterogeneous images containing distinct brightness at different parts to separate objects from the background, and employed watershed segmentation to distinguish touching cell objects. Both simulated studies and real-data analysis of large microscopy images demonstrate the scalability and accuracy of our approach compared with the alternatives.
Cell instance segmentation is a fundamental task in digital pathology with broad clinical applications. Recently, vision foundation models, which are predominantly based on Vision Transformers (ViTs), have achieved remarkable success in pathology image analysis. However, their improvements in cell instance segmentation remain limited. A key challenge arises from the tokenization process in ViTs, which substantially reduces the spatial resolution of input images, leading to suboptimal segmentation quality, especially for small and densely packed cells. To address this problem, we propose CellVTA (Cell Vision Transformer with Adapter), a novel method that improves the performance of vision foundation models for cell instance segmentation by incorporating a CNN-based adapter module. This adapter extracts high-resolution spatial information from input images and injects it into the ViT through a cross-attention mechanism. Our method preserves the core architecture of ViT, ensuring seamless integration with pretrained foundation models. Extensive experiments show that CellVTA achieves 0.538 mPQ on the CoNIC dataset and 0.506 mPQ on the PanNuke dataset, which significantly outperforms the state-of-the-art cell segmentation methods. Ablation studies confirm the superiority of our approach over other fine-tuning strategies, including decoder-only fine-tuning and full fine-tuning. Our code and models are publicly available at https://github.com/JieZheng-ShanghaiTech/CellVTA.
Cell instance segmentation (CIS) is crucial for identifying individual cell morphologies in histopathological images, providing valuable insights for biological and medical research. While unsupervised CIS (UCIS) models aim to reduce the heavy reliance on labor-intensive image annotations, they fail to accurately capture cell boundaries, causing missed detections and poor performance. Recognizing the absence of error-free instances as a key limitation, we present COIN (COnfidence score-guided INstance distillation), a novel annotation-free framework with three key steps: (1) Increasing the sensitivity for the presence of error-free instances via unsupervised semantic segmentation with optimal transport, leveraging its ability to discriminate spatially minor instances, (2) Instance-level confidence scoring to measure the consistency between model prediction and refined mask and identify highly confident instances, offering an alternative to ground truth annotations, and (3) Progressive expansion of confidence with recursive self-distillation. Extensive experiments across six datasets show COIN outperforming existing UCIS methods, even surpassing semi- and weakly-supervised approaches across all metrics on the MoNuSeg and TNBC datasets. The code is available at https://github.com/shjo-april/COIN.
This study introduces SEFI (SEgmentation-Free Integration), a novel method for integrating morphological features of cell nuclei with spatial transcriptomics data. Cell segmentation poses a significant challenge in the analysis of spatial transcriptomics data, as tissue-specific structural complexities and densely packed cells in certain regions make it difficult to develop a universal approach. SEFI addresses this by utilizing self-supervised learning to extract morphological features from fluorescent nuclear staining images, enhancing the clustering of gene expression data without requiring segmentation. We demonstrate SEFI on spatially resolved gene expression profiles of the developing retina, acquired using multiplexed single molecule Fluorescence In Situ Hybridization (smFISH). SEFI is publicly available at https://github.com/eduardchelebian/sefi.
Precise segmentation and classification of cell instances are vital for analyzing the tissue microenvironment in histology images, supporting medical diagnosis, prognosis, treatment planning, and studies of brain cytoarchitecture. However, the creation of high-quality annotated datasets for training remains a major challenge. This study introduces a novel single-stage approach (HistoSmith) for generating image-label pairs to augment histology datasets. Unlike state-of-the-art methods that utilize diffusion models with separate components for label and image generation, our approach employs a latent diffusion model to learn the joint distribution of cellular layouts, classification masks, and histology images. This model enables tailored data generation by conditioning on user-defined parameters such as cell types, quantities, and tissue types. Trained on the Conic H&E histopathology dataset and the Nissl-stained CytoDArk0 dataset, the model generates realistic and diverse labeled samples. Experimental results demonstrate improvements in cell instance segmentation and classification, particularly for underrepresented cell types like neutrophils in the Conic dataset. These findings underscore the potential of our approach to address data scarcity challenges.
Multi-class cell segmentation in high-resolution gigapixel whole slide images (WSI) is crucial for various clinical applications. However, training such models typically requires labor-intensive, pixel-wise annotations by domain experts. Recent efforts have democratized this process by involving lay annotators without medical expertise. However, conventional non-agent-based approaches struggle to handle annotation noise adaptively, as they lack mechanisms to mitigate false positives (FP) and false negatives (FN) at both the image-feature and pixel levels. In this paper, we propose a consensus-aware self-corrective AI agent that leverages the Consensus Matrix to guide its learning process. The Consensus Matrix defines regions where both the AI and annotators agree on cell and non-cell annotations, which are prioritized with stronger supervision. Conversely, areas of disagreement are adaptively weighted based on their feature similarity to high-confidence agreement regions, with more similar regions receiving greater attention. Additionally, contrastive learning is employed to separate features of noisy regions from those of reliable agreement regions by maximizing their dissimilarity. This paradigm enables the AI to iteratively refine noisy labels, enhancing its robustness. Validated on one real-world lay-annotated cell dataset and two simulated noisy datasets, our method demonstrates improved segmentation performance, effectively correcting FP and FN errors and showcasing its potential for training robust models on noisy datasets. The official implementation and cell annotations are publicly available at https://github.com/ddrrnn123/CASC-AI.
Recent advancements in foundation models have transformed computer vision, driving significant performance improvements across diverse domains, including digital histopathology. However, the advantages of domain-specific histopathology foundation models over general-purpose models for specialized tasks such as cell analysis remain underexplored. This study investigates the representation learning gap between these two categories by analyzing multi-level patch embeddings applied to cell instance segmentation and classification. We implement an encoder-decoder architecture with a consistent decoder and various encoders. These include convolutional, vision transformer (ViT), and hybrid encoders pre-trained on ImageNet-22K or LVD-142M, representing general-purpose foundation models. These are compared against ViT encoders from the recently released UNI, Virchow2, and Prov-GigaPath foundation models, trained on patches extracted from hundreds of thousands of histopathology whole-slide images. The decoder integrates patch embeddings from different encoder depths via skip connections to generate semantic and distance maps. These maps are then post-processed to create instance segmentation masks where each label corresponds to an individual cell and to perform cell-type classification. All encoders remain frozen during training to assess their pre-trained feature extraction capabilities. Using the PanNuke and CoNIC histopathology datasets, and the newly introduced Nissl-stained CytoDArk0 dataset for brain cytoarchitecture studies, we evaluate instance-level detection, segmentation accuracy, and cell-type classification. This study provides insights into the comparative strengths and limitations of general-purpose vs. histopathology foundation models, offering guidance for model selection in cell-focused histopathology and brain cytoarchitecture analysis workflows.
Epithelial cells form diverse structures from squamous spherical organoids to densely packed pseudostratified tissues. Quantification of cellular properties in these contexts requires high-resolution deep imaging and computational techniques to achieve truthful three-dimensional (3D) structural features. Here, we describe a detailed step-by-step protocol for sample preparation, imaging and deep-learning-assisted cell segmentation to achieve accurate quantification of fluorescently labelled individual cells in 3D within live tissues. We share the lessons learned through troubleshooting 3D imaging of Drosophila wing discs, including considerations on the choice of microscopy modality and settings (objective, sample mounting) and available segmentation methods. In addition, we include a computational pipeline alongside custom code to assist replication of the protocol. While we focus on the segmentation of cell outlines from membrane labelling, this protocol applies to a wide variety of samples, and we believe it be valuable for studying other tissues that demand complex analysis in 3D.
Digital Pathology is a cornerstone in the diagnosis and treatment of diseases. A key task in this field is the identification and segmentation of cells in hematoxylin and eosin-stained images. Existing methods for cell segmentation often require extensive annotated datasets for training and are limited to a predefined cell classification scheme. To overcome these limitations, we propose $\text{CellViT}^{{\scriptscriptstyle ++}}$, a framework for generalized cell segmentation in digital pathology. $\text{CellViT}^{{\scriptscriptstyle ++}}$ utilizes Vision Transformers with foundation models as encoders to compute deep cell features and segmentation masks simultaneously. To adapt to unseen cell types, we rely on a computationally efficient approach. It requires minimal data for training and leads to a drastically reduced carbon footprint. We demonstrate excellent performance on seven different datasets, covering a broad spectrum of cell types, organs, and clinical settings. The framework achieves remarkable zero-shot segmentation and data-efficient cell-type classification. Furthermore, we show that $\text{CellViT}^{{\scriptscriptstyle ++}}$ can leverage immunofluorescence stainings to generate training datasets without the need for pathologist annotations. The automated dataset generation approach surpasses the performance of networks trained on manually labeled data, demonstrating its effectiveness in creating high-quality training datasets without expert annotations. To advance digital pathology, $\text{CellViT}^{{\scriptscriptstyle ++}}$ is available as an open-source framework featuring a user-friendly, web-based interface for visualization and annotation. The code is available under https://github.com/TIO-IKIM/CellViT-plus-plus.
Recent trends in cell segmentation have shifted towards universal models to handle diverse cell morphologies and imaging modalities. However, for continuously emerging cell types and imaging techniques, these models still require hundreds or thousands of annotated cells for fine-tuning. We introduce CellSeg1, a practical solution for segmenting cells of arbitrary morphology and modality with a few dozen cell annotations in 1 image. By adopting Low-Rank Adaptation of the Segment Anything Model (SAM), we achieve robust cell segmentation. Tested on 19 diverse cell datasets, CellSeg1 trained on 1 image achieved 0.81 average mAP at 0.5 IoU, performing comparably to existing models trained on over 500 images. It also demonstrated superior generalization in cross-dataset tests on TissueNet. We found that high-quality annotation of a few dozen densely packed cells of varied sizes is key to effective segmentation. CellSeg1 provides an efficient solution for cell segmentation with minimal annotation effort.
Automated cell segmentation in microscopy images is essential for biomedical research, yet conventional methods are labor-intensive and prone to error. While deep learning-based approaches have proven effective, they often require large annotated datasets, which are scarce due to the challenges of manual annotation. To overcome this, we propose a novel framework for synthesizing densely annotated 2D and 3D cell microscopy images using cascaded diffusion models. Our method synthesizes 2D and 3D cell masks from sparse 2D annotations using multi-level diffusion models and NeuS, a 3D surface reconstruction approach. Following that, a pretrained 2D Stable Diffusion model is finetuned to generate realistic cell textures and the final outputs are combined to form cell populations. We show that training a segmentation model with a combination of our synthetic data and real data improves cell segmentation performance by up to 9\% across multiple datasets. Additionally, the FID scores indicate that the synthetic data closely resembles real data. The code for our proposed approach will be available at https://github.com/ruveydayilmaz0/cascaded_diffusion.
Advanced image segmentation and processing tools present an opportunity to study cell processes and their dynamics. However, image analysis is often routine and time-consuming. Nowadays, alternative data-driven approaches using deep learning are potentially offering automatized, accurate, and fast image analysis. In this paper, we extend the applications of Cellpose, a state-of-the-art cell segmentation framework, with feature extraction capabilities to assess morphological characteristics. We also introduce a dataset of DAPI and FITC stained cells to which our new method is applied.
Deep learning has revolutionized medical and biological imaging, particularly in segmentation tasks. However, segmenting biological cells remains challenging due to the high variability and complexity of cell shapes. Addressing this challenge requires high-quality datasets that accurately represent the diverse morphologies found in biological cells. Existing cell segmentation datasets are often limited by their focus on regular and uniform shapes. In this paper, we introduce a novel benchmark dataset of Ntera-2 (NT2) cells, a pluripotent carcinoma cell line, exhibiting diverse morphologies across multiple stages of differentiation, capturing the intricate and heterogeneous cellular structures that complicate segmentation tasks. To address these challenges, we propose an uncertainty-aware deep learning framework for complex cellular morphology segmentation (MorphoSeg) by incorporating sampling of virtual outliers from low-likelihood regions during training. Our comprehensive experimental evaluations against state-of-the-art baselines demonstrate that MorphoSeg significantly enhances segmentation accuracy, achieving up to a 7.74% increase in the Dice Similarity Coefficient (DSC) and a 28.36% reduction in the Hausdorff Distance. These findings highlight the effectiveness of our dataset and methodology in advancing cell segmentation capabilities, especially for complex and variable cell morphologies. The dataset and source code is publicly available at https://github.com/RanchoGoose/MorphoSeg.
Biomedical image segmentation is crucial for accurately diagnosing and analyzing various diseases. However, Convolutional Neural Networks (CNNs) and Transformers, the most commonly used architectures for this task, struggle to effectively capture long-range dependencies due to the inherent locality of CNNs and the computational complexity of Transformers. To address this limitation, we introduce TTT-Unet, a novel framework that integrates Test-Time Training (TTT) layers into the traditional U-Net architecture for biomedical image segmentation. TTT-Unet dynamically adjusts model parameters during the testing time, enhancing the model's ability to capture both local and long-range features. We evaluate TTT-Unet on multiple medical imaging datasets, including 3D abdominal organ segmentation in CT and MR images, instrument segmentation in endoscopy images, and cell segmentation in microscopy images. The results demonstrate that TTT-Unet consistently outperforms state-of-the-art CNN-based and Transformer-based segmentation models across all tasks. The code is available at https://github.com/rongzhou7/TTT-Unet.
Cell and nucleus segmentation are fundamental tasks for quantitative bioimage analysis. Despite progress in recent years, biologists and other domain experts still require novel algorithms to handle increasingly large and complex real-world datasets. These algorithms must not only achieve state-of-the-art accuracy, but also be optimized for efficiency, portability and user-friendliness. Here, we introduce InstanSeg: a novel embedding-based instance segmentation pipeline designed to identify cells and nuclei in microscopy images. Using six public cell segmentation datasets, we demonstrate that InstanSeg can significantly improve accuracy when compared to the most widely used alternative methods, while reducing the processing time by at least 60%. Furthermore, InstanSeg is designed to be fully serializable as TorchScript and supports GPU acceleration on a range of hardware. We provide an open-source implementation of InstanSeg in Python, in addition to a user-friendly, interactive QuPath extension for inference written in Java. Our code and pre-trained models are available at https://github.com/instanseg/instanseg .
In pathology, accurate and efficient analysis of Hematoxylin and Eosin (H\&E) slides is crucial for timely and effective cancer diagnosis. Although many deep learning solutions for nuclei instance segmentation and classification exist in the literature, they often entail high computational costs and resource requirements, thus limiting their practical usage in medical applications. To address this issue, we introduce a novel convolutional neural network, NuLite, a U-Net-like architecture designed explicitly on Fast-ViT, a state-of-the-art (SOTA) lightweight CNN. We obtained three versions of our model, NuLite-S, NuLite-M, and NuLite-H, trained on the PanNuke dataset. The experimental results prove that our models equal CellViT (SOTA) in terms of panoptic quality and detection. However, our lightest model, NuLite-S, is 40 times smaller in terms of parameters and about 8 times smaller in terms of GFlops, while our heaviest model is 17 times smaller in terms of parameters and about 7 times smaller in terms of GFlops. Moreover, our model is up to about 8 times faster than CellViT. Lastly, to prove the effectiveness of our solution, we provide a robust comparison of external datasets, namely CoNseP, MoNuSeg, and GlySAC. Our model is publicly available at https://github.com/CosmoIknosLab/NuLite
The segmentation of cell nuclei in tissue images stained with the blood dye hematoxylin and eosin (H$\&$E) is essential for various clinical applications and analyses. Due to the complex characteristics of cellular morphology, a large receptive field is considered crucial for generating high-quality segmentation. However, previous methods face challenges in achieving a balance between the receptive field and computational burden. To address this issue, we propose LKCell, a high-accuracy and efficient cell segmentation method. Its core insight lies in unleashing the potential of large convolution kernels to achieve computationally efficient large receptive fields. Specifically, (1) We transfer pre-trained large convolution kernel models to the medical domain for the first time, demonstrating their effectiveness in cell segmentation. (2) We analyze the redundancy of previous methods and design a new segmentation decoder based on large convolution kernels. It achieves higher performance while significantly reducing the number of parameters. We evaluate our method on the most challenging benchmark and achieve state-of-the-art results (0.5080 mPQ) in cell nuclei instance segmentation with only 21.6% FLOPs compared with the previous leading method. Our source code and models are available at https://github.com/hustvl/LKCell.
Early detection and accurate diagnosis can predict the risk of malignant disease transformation, thereby increasing the probability of effective treatment. Identifying mild syndrome with small pathological regions serves as an ominous warning and is fundamental in the early diagnosis of diseases. While deep learning algorithms, particularly convolutional neural networks (CNNs), have shown promise in segmenting medical objects, analyzing small areas in medical images remains challenging. This difficulty arises due to information losses and compression defects from convolution and pooling operations in CNNs, which become more pronounced as the network deepens, especially for small medical objects. To address these challenges, we propose a novel scale-variant attention-based network (SvANet) for accurately segmenting small-scale objects in medical images. The SvANet consists of scale-variant attention, cross-scale guidance, Monte Carlo attention, and vision transformer, which incorporates cross-scale features and alleviates compression artifacts for enhancing the discrimination of small medical objects. Quantitative experimental results demonstrate the superior performance of SvANet, achieving 96.12%, 96.11%, 89.79%, 84.15%, 80.25%, 73.05%, and 72.58% in mean Dice coefficient for segmenting kidney tumors, skin lesions, hepatic tumors, polyps, surgical excision cells, retinal vasculatures, and sperms, which occupy less than 1% of the image areas in KiTS23, ISIC 2018, ATLAS, PolypGen, TissueNet, FIVES, and SpermHealth datasets, respectively.
Accurate segmentation of medical images is crucial for diagnostic purposes, including cell segmentation, tumor identification, and organ localization. Traditional convolutional neural network (CNN)-based approaches struggled to achieve precise segmentation results due to their limited receptive fields, particularly in cases involving multi-organ segmentation with varying shapes and sizes. The transformer-based approaches address this limitation by leveraging the global receptive field, but they often face challenges in capturing local information required for pixel-precise segmentation. In this work, we introduce DwinFormer, a hierarchical encoder-decoder architecture for medical image segmentation comprising a directional window (Dwin) attention and global self-attention (GSA) for feature encoding. The focus of our design is the introduction of Dwin block within DwinFormer that effectively captures local and global information along the horizontal, vertical, and depthwise directions of the input feature map by separately performing attention in each of these directional volumes. To this end, our Dwin block introduces a nested Dwin attention (NDA) that progressively increases the receptive field in horizontal, vertical, and depthwise directions and a convolutional Dwin attention (CDA) that captures local contextual information for the attention computation. While the proposed Dwin block captures local and global dependencies at the first two high-resolution stages of DwinFormer, the GSA block encodes global dependencies at the last two lower-resolution stages. Experiments over the challenging 3D Synapse Multi-organ dataset and Cell HMS dataset demonstrate the benefits of our DwinFormer over the state-of-the-art approaches. Our source code will be publicly available at \url{https://github.com/Daniyanaj/DWINFORMER}.
Deep Learning (DL) models have been successfully applied to many applications including biomedical cell segmentation and classification in histological images. These models require large amounts of annotated data which might not always be available, especially in the medical field where annotations are scarce and expensive. To overcome this limitation, we propose a novel pipeline for generating synthetic datasets for cell segmentation. Given only a handful of annotated images, our method generates a large dataset of images which can be used to effectively train DL instance segmentation models. Our solution is designed to generate cells of realistic shapes and placement by allowing experts to incorporate domain knowledge during the generation of the dataset.