Scarcity of annotated data, particularly for rare or atypical morphologies, present significant challenges for cell and nuclei segmentation in computational pathology. While manual annotation is labor-intensive and costly, synthetic data offers a cost-effective alternative. We introduce a Multimodal Semantic Diffusion Model (MSDM) for generating realistic pixel-precise image-mask pairs for cell and nuclei segmentation. By conditioning the generative process with cellular/nuclear morphologies (using horizontal and vertical maps), RGB color characteristics, and BERT-encoded assay/indication metadata, MSDM generates datasests with desired morphological properties. These heterogeneous modalities are integrated via multi-head cross-attention, enabling fine-grained control over the generated images. Quantitative analysis demonstrates that synthetic images closely match real data, with low Wasserstein distances between embeddings of generated and real images under matching biological conditions. The incorporation of these synthetic samples, exemplified by columnar cells, significantly improves segmentation model accuracy on columnar cells. This strategy systematically enriches data sets, directly targeting model deficiencies. We highlight the effectiveness of multimodal diffusion-based augmentation for advancing the robustness and generalizability of cell and nuclei segmentation models. Thereby, we pave the way for broader application of generative models in computational pathology.

While multiplex immunofluorescence (mIF) imaging provides deep, spatially-resolved molecular data, integrating this information with the morphological standard of Hematoxylin & Eosin (H&E) can be very important for obtaining complementary information about the underlying tissue. Generating a virtual H&E stain from mIF data offers a powerful solution, providing immediate morphological context. Crucially, this approach enables the application of the vast ecosystem of H&E-based computer-aided diagnosis (CAD) tools to analyze rich molecular data, bridging the gap between molecular and morphological analysis. In this work, we investigate the use of a multi-level Vector-Quantized Generative Adversarial Network (VQGAN) to create high-fidelity virtual H&E stains from mIF images. We rigorously evaluated our VQGAN against a standard conditional GAN (cGAN) baseline on two publicly available colorectal cancer datasets, assessing performance on both image similarity and functional utility for downstream analysis. Our results show that while both architectures produce visually plausible images, the virtual stains generated by our VQGAN provide a more effective substrate for computer-aided diagnosis. Specifically, downstream nuclei segmentation and semantic preservation in tissue classification tasks performed on VQGAN-generated images demonstrate superior performance and agreement with ground-truth analysis compared to those from the cGAN. This work establishes that a multi-level VQGAN is a robust and superior architecture for generating scientifically useful virtual stains, offering a viable pathway to integrate the rich molecular data of mIF into established and powerful H&E-based analytical workflows.

Prof. Dr. Bilge Ayca Kirmizi, akarabork@yahoo.com 1 Introduction Breast cancer is the most frequently diagnosed form of cancer and is the second leading cause of death caused by cancer in women. In order to diagnose breast cancer type, stage, and grade accurately, examination of tissue biopsies and operation specimens is necessary. The biopsy specimens must be fixed embedded in paraffin blocks, mounted on glass slides and stained. Hematoxylin and Eosin (H&E), is a routine stain used in pathology laboratories all over the globe, which gives a good contrast of a tissue section and is commonly used to identify nuclei and cytoplasm [1]. Nevertheless, histopathological examination of the prapared slides involves laborious, time - consuming processes that are limited by specimen quality and pathologist experience.

Segmentation of nuclei regions from histological images is an important task for automated computer-aided analysis of histological images, particularly in the presence of impermissible color variation in the color appearance of stained tissue images. While color normalization enables better nuclei segmentation, accurate segmentation of nuclei structures makes color normalization rather trivial. In this respect, the paper proposes a novel deep generative model for simultaneously segmenting nuclei structures and normalizing color appearance of stained histological images.This model judiciously integrates the merits of truncated normal distribution and spatial attention. The model assumes that the latent color appearance information, corresponding to a particular histological image, is independent of respective nuclei segmentation map as well as embedding map information. The disentangled representation makes the model generalizable and adaptable as the modification or loss in color appearance information cannot be able to affect the nuclei segmentation map as well as embedding information. Also, for dealing with the stain overlap of associated histochemical reagents, the prior for latent color appearance code is assumed to be a mixture of truncated normal distributions. The proposed model incorporates the concept of spatial attention for segmentation of nuclei regions from histological images. The performance of the proposed approach, along with a comparative analysis with related state-of-the-art algorithms, has been demonstrated on publicly available standard histological image data sets.

Segmentation of nuclei regions from histological images enables morphometric analysis of nuclei structures, which in turn helps in the detection and diagnosis of diseases under consideration. To develop a nuclei segmentation algorithm, applicable to different types of target domain representations, image-to-image translation networks can be considered as they are invariant to target domain image representations. One of the important issues with image-to-image translation models is that they fail miserably when the information content between two image domains are asymmetric in nature. In this regard, the paper introduces a new deep generative model for segmenting nuclei structures from histological images. The proposed model considers an embedding space for handling information-disparity between information-rich histological image space and information-poor segmentation map domain. Integrating judiciously the concepts of optimal transport and measure theory, the model develops an invertible generator, which provides an efficient optimization framework with lower network complexity. The concept of invertible generator automatically eliminates the need of any explicit cycle-consistency loss. The proposed model also introduces a spatially-constrained squeeze operation within the framework of invertible generator to maintain spatial continuity within the image patches. The model provides a better trade-off between network complexity and model performance compared to other existing models having complex network architectures. The performance of the proposed deep generative model, along with a comparison with state-of-the-art nuclei segmentation methods, is demonstrated on publicly available histological image data sets.

Digital pathology, augmented by artificial intelligence (AI), holds significant promise for improving the workflow of pathologists. However, challenges such as the labor-intensive annotation of whole slide images (WSIs), high computational demands, and trust concerns arising from the absence of uncertainty estimation in predictions hinder the practical application of current AI methodologies in histopathology. To address these issues, we present a novel trustful fully unsupervised multi-level segmentation methodology (TUMLS) for WSIs. TUMLS adopts an autoencoder (AE) as a feature extractor to identify the different tissue types within low-resolution training data. It selects representative patches from each identified group based on an uncertainty measure and then does unsupervised nuclei segmentation in their respective higher-resolution space without using any ML algorithms. Crucially, this solution integrates seamlessly into clinicians workflows, transforming the examination of a whole WSI into a review of concise, interpretable cross-level insights. This integration significantly enhances and accelerates the workflow while ensuring transparency. We evaluated our approach using the UPENN-GBM dataset, where the AE achieved a mean squared error (MSE) of 0.0016. Additionally, nucleus segmentation is assessed on the MoNuSeg dataset, outperforming all unsupervised approaches with an F1 score of 77.46% and a Jaccard score of 63.35%. These results demonstrate the efficacy of TUMLS in advancing the field of digital pathology.

In histological pathology, frozen sections are often used for rapid diagnosis during surgeries, as they can be produced within minutes. However, they suffer from artifacts and often lack crucial diagnostic details, particularly within the cell nuclei region. Permanent sections, on the other hand, contain more diagnostic detail but require a time-intensive preparation process. Here, we present a generative deep learning approach to enhance frozen section images by leveraging guidance from permanent sections. Our method places a strong emphasis on the nuclei region, which contains critical information in both frozen and permanent sections. Importantly, our approach avoids generating artificial data in blank regions, ensuring that the network only enhances existing features without introducing potentially unreliable information. We achieve this through a segmented attention network, incorporating nuclei-segmented images during training and adding an additional loss function to refine the nuclei details in the generated permanent images. We validated our method across various tissues, including kidney, breast, and colon. This approach significantly improves histological efficiency and diagnostic accuracy, enhancing frozen section images within seconds, and seamlessly integrating into existing laboratory workflows.

Training AI foundation models has emerged as a promising large-scale learning approach for addressing real-world healthcare challenges, including digital pathology. While many of these models have been developed for tasks like disease diagnosis and tissue quantification using extensive and diverse training datasets, their readiness for deployment on some arguably simplest tasks, such as nuclei segmentation within a single organ (e.g., the kidney), remains uncertain. This paper seeks to answer this key question, "How good are we?", by thoroughly evaluating the performance of recent cell foundation models on a curated multi-center, multi-disease, and multi-species external testing dataset. Additionally, we tackle a more challenging question, "How can we improve?", by developing and assessing human-in-the-loop data enrichment strategies aimed at enhancing model performance while minimizing the reliance on pixel-level human annotation. To address the first question, we curated a multicenter, multidisease, and multispecies dataset consisting of 2,542 kidney whole slide images (WSIs). Three state-of-the-art (SOTA) cell foundation models-Cellpose, StarDist, and CellViT-were selected for evaluation. To tackle the second question, we explored data enrichment algorithms by distilling predictions from the different foundation models with a human-in-the-loop framework, aiming to further enhance foundation model performance with minimal human efforts. Our experimental results showed that all three foundation models improved over their baselines with model fine-tuning with enriched data. Interestingly, the baseline model with the highest F1 score does not yield the best segmentation outcomes after fine-tuning. This study establishes a benchmark for the development and deployment of cell vision foundation models tailored for real-world data applications.

Accurate nuclei segmentation is an essential foundation for various applications in computational pathology, including cancer diagnosis and treatment planning. Even slight variations in nuclei representations can significantly impact these downstream tasks. However, achieving accurate segmentation remains challenging due to factors like clustered nuclei, high intra-class variability in size and shape, resemblance to other cells, and color or contrast variations between nuclei and background. Despite the extensive utilization of Convolutional Neural Networks (CNNs) in medical image segmentation, they may have trouble capturing long-range dependencies crucial for accurate nuclei delineation. Transformers address this limitation but might miss essential low-level features. To overcome these limitations, we utilized CNN-Transformer-based techniques for nuclei segmentation in H&E stained histology images. In this work, we proposed two CNN-Transformer architectures, Nuclei Hybrid Vision Transformer (NucleiHVT) and Channel Boosted Nuclei Hybrid Vision Transformer (CB-NucleiHVT), that leverage the strengths of both CNNs and Transformers to effectively learn nuclei boundaries in multi-organ histology images. The first architecture, NucleiHVT is inspired by the UNet architecture and incorporates the dual attention mechanism to capture both multi-level and multi-scale context effectively. The CB-NucleiHVT network, on the other hand, utilizes the concept of channel boosting to learn diverse feature spaces, enhancing the model's ability to distinguish subtle variations in nuclei characteristics. Detailed evaluation of two medical image segmentation datasets shows that the proposed architectures outperform existing CNN-based, Transformer-based, and hybrid methods. The proposed networks demonstrated effective results both in terms of quantitative metrics, and qualitative visual assessment.

Deep learning has achieved impressive results in nuclei segmentation, but the massive requirement for pixel-wise labels remains a significant challenge. To alleviate the annotation burden, existing methods generate pseudo masks for model training using point labels. However, the generated masks are inevitably different from the ground truth, and these dissimilarities are not handled reasonably during the network training, resulting in the subpar performance of the segmentation model. To tackle this issue, we propose a framework named DoNuSeg, enabling \textbf{D}ynamic pseudo label \textbf{O}ptimization in point-supervised \textbf{Nu}clei \textbf{Seg}mentation. Specifically, DoNuSeg takes advantage of class activation maps (CAMs) to adaptively capture regions with semantics similar to annotated points. To leverage semantic diversity in the hierarchical feature levels, we design a dynamic selection module to choose the optimal one among CAMs from different encoder blocks as pseudo masks. Meanwhile, a CAM-guided contrastive module is proposed to further enhance the accuracy of pseudo masks. In addition to exploiting the semantic information provided by CAMs, we consider location priors inherent to point labels, developing a task-decoupled structure for effectively differentiating nuclei. Extensive experiments demonstrate that DoNuSeg outperforms state-of-the-art point-supervised methods. The code is available at https://github.com/shinning0821/MICCAI24-DoNuSeg.