The digitization of histology slides has revolutionized pathology, providing massive datasets for cancer diagnosis and research. Contrastive self-supervised and vision-language models have been shown to effectively mine large pathology datasets to learn discriminative representations. On the other hand, generative models, capable of synthesizing realistic and diverse images, present a compelling solution to address unique problems in pathology that involve synthesizing images; overcoming annotated data scarcity, enabling privacy-preserving data sharing, and performing inherently generative tasks, such as virtual staining. We introduce PixCell, the first diffusion-based generative foundation model for histopathology. We train PixCell on PanCan-30M, a vast, diverse dataset derived from 69,184 H\&E-stained whole slide images covering various cancer types. We employ a progressive training strategy and a self-supervision-based conditioning that allows us to scale up training without any annotated data. PixCell generates diverse and high-quality images across multiple cancer types, which we find can be used in place of real data to train a self-supervised discriminative model. Synthetic images shared between institutions are subject to fewer regulatory barriers than would be the case with real clinical images. Furthermore, we showcase the ability to precisely control image generation using a small set of annotated images, which can be used for both data augmentation and educational purposes. Testing on a cell segmentation task, a mask-guided PixCell enables targeted data augmentation, improving downstream performance. Finally, we demonstrate PixCell's ability to use H\&E structural staining to infer results from molecular marker studies; we use this capability to infer IHC staining from H\&E images. Our trained models are publicly released to accelerate research in computational pathology.
The principle of translation equivariance (if an input image is translated an output image should be translated by the same amount), led to the development of convolutional neural networks that revolutionized machine vision. Other symmetries, like rotations and reflections, play a similarly critical role, especially in biomedical image analysis, but exploiting these symmetries has not seen wide adoption. We hypothesize that this is partially due to the mathematical complexity of methods used to exploit these symmetries, which often rely on representation theory, a bespoke concept in differential geometry and group theory. In this work, we show that the same equivariance can be achieved using a simple form of convolution kernels that we call ``moment kernels,'' and prove that all equivariant kernels must take this form. These are a set of radially symmetric functions of a spatial position $x$, multiplied by powers of the components of $x$ or the identity matrix. We implement equivariant neural networks using standard convolution modules, and provide architectures to execute several biomedical image analysis tasks that depend on equivariance principles: classification (outputs are invariant under orthogonal transforms), 3D image registration (outputs transform like a vector), and cell segmentation (quadratic forms defining ellipses transform like a matrix).
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.
Medical AI assistants support doctors in disease diagnosis, medical image analysis, and report generation. However, they still face significant challenges in clinical use, including limited accuracy with multimodal content and insufficient validation in real-world settings. We propose RCMed, a full-stack AI assistant that improves multimodal alignment in both input and output, enabling precise anatomical delineation, accurate localization, and reliable diagnosis through hierarchical vision-language grounding. A self-reinforcing correlation mechanism allows visual features to inform language context, while language semantics guide pixel-wise attention, forming a closed loop that refines both modalities. This correlation is enhanced by a color region description strategy, translating anatomical structures into semantically rich text to learn shape-location-text relationships across scales. Trained on 20 million image-mask-description triplets, RCMed achieves state-of-the-art precision in contextualizing irregular lesions and subtle anatomical boundaries, excelling in 165 clinical tasks across 9 modalities. It achieved a 23.5% relative improvement in cell segmentation from microscopy images over prior methods. RCMed's strong vision-language alignment enables exceptional generalization, with state-of-the-art performance in external validation across 20 clinically significant cancer types, including novel tasks. This work demonstrates how integrated multimodal models capture fine-grained patterns, enabling human-level interpretation in complex scenarios and advancing human-centric AI healthcare.
With the rapid development of microscopy imaging technology, the requirement for robust segmentation and quantification of cells or nanoparticles increases greatly. It remains challenging due to the diversity of the cell or nanoparticle types, the arbitrary shapes, and the large numbers of cells or nanoparticles. The most existing methods are only capable of segmenting some specific types of cells or nanoparticles. In this paper, we propose a more versatile approach that is capable of segmenting a variety of cells or nanoparticles. It consists of five parts: 1) automatic gradient image formation; 2) automatic threshold selection; 3) manual calibration of the threshold selection method for each specific type of cell or nanoparticle images; 4) manual determination of the segmentation cases for each specific type of cell or nanoparticle images; and 5) automatic quantification by iterative morphological erosion. After the parameter, N is calibrated and the segmentation case is determined manually for each specific type of cell or nanoparticle images with one or several typical images; only parts 1), 2), and 5) are needed for the rest of processing and they are automatic. The proposed approach is tested with different types of cell and nanoparticle images. Experimental results verified its effectiveness.
Pap smear image segmentation is crucial for cervical cancer diagnosis. However, traditional segmentation models often struggle with complex cellular structures and variations in pap smear images. This study proposes a hybrid Dense-UNet201 optimization approach that integrates a pretrained DenseNet201 as the encoder for the U-Net architecture and optimizes it using the spider monkey optimization (SMO) algorithm. The Dense-UNet201 model excelled at feature extraction. The SMO was modified to handle categorical and discrete parameters. The SIPaKMeD dataset was used in this study and evaluated using key performance metrics, including loss, accuracy, Intersection over Union (IoU), and Dice coefficient. The experimental results showed that Dense-UNet201 outperformed U-Net, Res-UNet50, and Efficient-UNetB0. SMO Dense-UNet201 achieved a segmentation accuracy of 96.16%, an IoU of 91.63%, and a Dice coefficient score of 95.63%. These findings underscore the effectiveness of image preprocessing, pretrained models, and metaheuristic optimization in improving medical image analysis and provide new insights into cervical cell segmentation methods.
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.
Producing high-quality segmentation masks for medical images is a fundamental challenge in biomedical image analysis. Recent research has explored large-scale supervised training to enable segmentation across various medical imaging modalities and unsupervised training to facilitate segmentation without dense annotations. However, constructing a model capable of segmenting diverse medical images in a zero-shot manner without any annotations remains a significant hurdle. This paper introduces the Attention Diffusion Zero-shot Unsupervised System (ADZUS), a novel approach that leverages self-attention diffusion models for zero-shot biomedical image segmentation. ADZUS harnesses the intrinsic capabilities of pre-trained diffusion models, utilizing their generative and discriminative potentials to segment medical images without requiring annotated training data or prior domain-specific knowledge. The ADZUS architecture is detailed, with its integration of self-attention mechanisms that facilitate context-aware and detail-sensitive segmentations being highlighted. Experimental results across various medical imaging datasets, including skin lesion segmentation, chest X-ray infection segmentation, and white blood cell segmentation, reveal that ADZUS achieves state-of-the-art performance. Notably, ADZUS reached Dice scores ranging from 88.7\% to 92.9\% and IoU scores from 66.3\% to 93.3\% across different segmentation tasks, demonstrating significant improvements in handling novel, unseen medical imagery. It is noteworthy that while ADZUS demonstrates high effectiveness, it demands substantial computational resources and extended processing times. The model's efficacy in zero-shot settings underscores its potential to reduce reliance on costly annotations and seamlessly adapt to new medical imaging tasks, thereby expanding the diagnostic capabilities of AI-driven medical imaging technologies.
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.
3D cell segmentation methods are often hindered by \emph{oversegmentation}, where a single cell is incorrectly split into multiple fragments. This degrades the final segmentation quality and is notoriously difficult to resolve, as oversegmentation errors often resemble \emph{natural gaps} between adjacent cells. Our work makes two key contributions. First, for 3D cell segmentation, we are the first work to formulate oversegmentation as a concrete problem and propose a geometric framework to identify and correct these errors. Our approach builds a pre-trained classifier using both 2D geometric and 3D topological features extracted from flawed 3D segmentation results. Second, we introduce a novel metric, \emph{Geo-Wasserstein} divergence, to quantify changes in 2D geometries. This captures the evolving trends in cell mask shape changes in a geometry-aware manner. We validate our method through extensive experiments on in-domain plant datasets, including both synthesized and real cases, as well as on out-of-domain animal datasets to demonstrate transfer learning performance. An ablation study further highlights the contribution of the \emph{Geo-Wasserstein} divergence. A clear pipeline is provided for end-users to build pre-trained models to any labeled dataset.
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.
Pathological cell semantic segmentation is a fundamental technology in computational pathology, essential for applications like cancer diagnosis and effective treatment. Given that multiple cell types exist across various organs, with subtle differences in cell size and shape, multi-organ, multi-class cell segmentation is particularly challenging. Most existing methods employ multi-branch frameworks to enhance feature extraction, but often result in complex architectures. Moreover, reliance on visual information limits performance in multi-class analysis due to intricate textural details. To address these challenges, we propose a Multi-OrgaN multi-Class cell semantic segmentation method with a single brancH (MONCH) that leverages vision-language input. Specifically, we design a hierarchical feature extraction mechanism to provide coarse-to-fine-grained features for segmenting cells of various shapes, including high-frequency, convolutional, and topological features. Inspired by the synergy of textual and multi-grained visual features, we introduce a progressive prompt decoder to harmonize multimodal information, integrating features from fine to coarse granularity for better context capture. Extensive experiments on the PanNuke dataset, which has significant class imbalance and subtle cell size and shape variations, demonstrate that MONCH outperforms state-of-the-art cell segmentation methods and vision-language models. Codes and implementations will be made publicly available.
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.
Multiplex Imaging (MI) enables the simultaneous visualization of multiple biological markers in separate imaging channels at subcellular resolution, providing valuable insights into cell-type heterogeneity and spatial organization. However, current computational pipelines rely on cell segmentation algorithms, which require laborious fine-tuning and can introduce downstream errors due to inaccurate single-cell representations. We propose a segmentation-free deep learning approach that leverages grouped convolutions to learn interpretable embedded features from each imaging channel, enabling robust cell-type identification without manual feature selection. Validated on an Imaging Mass Cytometry dataset of 1.8 million cells from neuroblastoma patients, our method enables the accurate identification of known cell types, showcasing its scalability and suitability for high-dimensional MI data.