Immunohistochemical (IHC) virtual staining is a task that generates virtual IHC images from H\&E images while maintaining pathological semantic consistency with adjacent slices. This task aims to achieve cross-domain mapping between morphological structures and staining patterns through generative models, providing an efficient and cost-effective solution for pathological analysis. However, under weakly paired conditions, spatial heterogeneity between adjacent slices presents significant challenges. This can lead to inaccurate one-to-many mappings and generate results that are inconsistent with the pathological semantics of adjacent slices. To address this issue, we propose a novel unbalanced self-information feature transport for IHC virtual staining, named USIGAN, which extracts global morphological semantics without relying on positional correspondence.By removing weakly paired terms in the joint marginal distribution, we effectively mitigate the impact of weak pairing on joint distributions, thereby significantly improving the content consistency and pathological semantic consistency of the generated results. Moreover, we design the Unbalanced Optimal Transport Consistency (UOT-CTM) mechanism and the Pathology Self-Correspondence (PC-SCM) mechanism to construct correlation matrices between H\&E and generated IHC in image-level and real IHC and generated IHC image sets in intra-group level.. Experiments conducted on two publicly available datasets demonstrate that our method achieves superior performance across multiple clinically significant metrics, such as IoD and Pearson-R correlation, demonstrating better clinical relevance.
Virtual staining is a promising technique that uses deep generative models to recreate histological stains, providing a faster and more cost-effective alternative to traditional tissue chemical staining. Specifically for H&E-HER2 staining transfer, despite a rising trend in publications, the lack of sufficient public datasets has hindered progress in the topic. Additionally, it is currently unclear which model frameworks perform best for this particular task. In this paper, we introduce the HER2match dataset, the first publicly available dataset with the same breast cancer tissue sections stained with both H&E and HER2. Furthermore, we compare the performance of several Generative Adversarial Networks (GANs) and Diffusion Models (DMs), and implement a novel Brownian Bridge Diffusion Model for H&E-HER2 translation. Our findings indicate that, overall, GANs perform better than DMs, with only the BBDM achieving comparable results. Furthermore, we emphasize the importance of data alignment, as all models trained on HER2match produced vastly improved visuals compared to the widely used consecutive-slide BCI dataset. This research provides a new high-quality dataset ([available upon publication acceptance]), improving both model training and evaluation. In addition, our comparison of frameworks offers valuable guidance for researchers working on the topic.
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.
Comprehensive assessment of the various aspects of the brain's microstructure requires the use of complementary imaging techniques. This includes measuring the spatial distribution of cell bodies (cytoarchitecture) and nerve fibers (myeloarchitecture). The gold standard for cytoarchitectonic analysis is light microscopic imaging of cell-body stained tissue sections. To reveal the 3D orientations of nerve fibers, 3D Polarized Light Imaging (3D-PLI) has been introduced as a reliable technique providing a resolution in the micrometer range while allowing processing of series of complete brain sections. 3D-PLI acquisition is label-free and allows subsequent staining of sections after measurement. By post-staining for cell bodies, a direct link between fiber- and cytoarchitecture can potentially be established within the same section. However, inevitable distortions introduced during the staining process make a nonlinear and cross-modal registration necessary in order to study the detailed relationships between cells and fibers in the images. In addition, the complexity of processing histological sections for post-staining only allows for a limited number of samples. In this work, we take advantage of deep learning methods for image-to-image translation to generate a virtual staining of 3D-PLI that is spatially aligned at the cellular level. In a supervised setting, we build on a unique dataset of brain sections, to which Cresyl violet staining has been applied after 3D-PLI measurement. To ensure high correspondence between both modalities, we address the misalignment of training data using Fourier-based registration methods. In this way, registration can be efficiently calculated during training for local image patches of target and predicted staining. We demonstrate that the proposed method enables prediction of a Cresyl violet staining from 3D-PLI, matching individual cell instances.
Hematoxylin and eosin (H&E) staining is a gold standard for microscopic diagnosis in pathology. However, H&E staining does not capture all the diagnostic information that may be needed. To obtain additional molecular information, immunohistochemical (IHC) stains highlight proteins that mark specific cell types, such as CD3 for T-cells or CK8/18 for epithelial cells. While IHC stains are vital for prognosis and treatment guidance, they are typically only available at specialized centers and time consuming to acquire, leading to treatment delays for patients. Virtual staining, enabled by deep learning-based image translation models, provides a promising alternative by computationally generating IHC stains from H&E stained images. Although many GAN and diffusion based image to image (I2I) translation methods have been used for virtual staining, these models treat image patches as independent data points, which results in increased and more diverse data requirements for effective generation. We present ImplicitStainer, a novel approach that leverages local implicit functions to improve image translation, specifically virtual staining performance, by focusing on pixel-level predictions. This method enhances robustness to variations in dataset sizes, delivering high-quality results even with limited data. We validate our approach on two datasets using a comprehensive set of metrics and benchmark it against over fifteen state-of-the-art GAN- and diffusion based models. Full Code and models trained will be released publicly via Github upon acceptance.
In histopathology, tissue sections are typically stained using common H&E staining or special stains (MAS, PAS, PASM, etc.) to clearly visualize specific tissue structures. The rapid advancement of deep learning offers an effective solution for generating virtually stained images, significantly reducing the time and labor costs associated with traditional histochemical staining. However, a new challenge arises in separating the fundamental visual characteristics of tissue sections from the visual differences induced by staining agents. Additionally, virtual staining often overlooks essential pathological knowledge and the physical properties of staining, resulting in only style-level transfer. To address these issues, we introduce, for the first time in virtual staining tasks, a pathological vision-language large model (VLM) as an auxiliary tool. We integrate contrastive learnable prompts, foundational concept anchors for tissue sections, and staining-specific concept anchors to leverage the extensive knowledge of the pathological VLM. This approach is designed to describe, frame, and enhance the direction of virtual staining. Furthermore, we have developed a data augmentation method based on the constraints of the VLM. This method utilizes the VLM's powerful image interpretation capabilities to further integrate image style and structural information, proving beneficial in high-precision pathological diagnostics. Extensive evaluations on publicly available multi-domain unpaired staining datasets demonstrate that our method can generate highly realistic images and enhance the accuracy of downstream tasks, such as glomerular detection and segmentation. Our code is available at: https://github.com/CZZZZZZZZZZZZZZZZZ/VPGAN-HARBOR
Polarization, as a new optical imaging tool, has been explored to assist in the diagnosis of pathology. Moreover, converting the polarimetric Mueller Matrix (MM) to standardized stained images becomes a promising approach to help pathologists interpret the results. However, existing methods for polarization-based virtual staining are still in the early stage, and the diffusion-based model, which has shown great potential in enhancing the fidelity of the generated images, has not been studied yet. In this paper, a Regulated Bridge Diffusion Model (RBDM) for polarization-based virtual staining is proposed. RBDM utilizes the bidirectional bridge diffusion process to learn the mapping from polarization images to other modalities such as H\&E and fluorescence. And to demonstrate the effectiveness of our model, we conduct the experiment on our manually collected dataset, which consists of 18,000 paired polarization, fluorescence and H\&E images, due to the unavailability of the public dataset. The experiment results show that our model greatly outperforms other benchmark methods. Our dataset and code will be released upon acceptance.
The emergence of virtual staining technology provides a rapid and efficient alternative for researchers in tissue pathology. It enables the utilization of unlabeled microscopic samples to generate virtual replicas of chemically stained histological slices, or facilitate the transformation of one staining type into another. The remarkable performance of generative networks, such as CycleGAN, offers an unsupervised learning approach for virtual coloring, overcoming the limitations of high-quality paired data required in supervised learning. Nevertheless, large-scale color transformation necessitates processing large field-of-view images in patches, often resulting in significant boundary inconsistency and artifacts. Additionally, the transformation between different colorized modalities typically needs further efforts to modify loss functions and tune hyperparameters for independent training of networks. In this study, we introduce a general virtual staining framework that is adaptable to various conditions. We propose a loss function based on the value mapping constraint to ensure the accuracy of virtual coloring between different pathological modalities, termed the Value Mapping Generative Adversarial Network (VM-GAN). Meanwhile, we present a confidence-based tiling method to address the challenge of boundary inconsistency arising from patch-wise processing. Experimental results on diverse data with varying staining protocols demonstrate that our method achieves superior quantitative indicators and improved visual perception.
Virtual staining leverages computer-aided techniques to transfer the style of histochemically stained tissue samples to other staining types. In virtual staining of pathological images, maintaining strict structural consistency is crucial, as these images emphasize structural integrity more than natural images. Even slight structural alterations can lead to deviations in diagnostic semantic information. Furthermore, the unpaired characteristic of virtual staining data may compromise the preservation of pathological diagnostic content. To address these challenges, we propose a dual-path inversion virtual staining method using prompt learning, which optimizes visual prompts to control content and style, while preserving complete pathological diagnostic content. Our proposed inversion technique comprises two key components: (1) Dual Path Prompted Strategy, we utilize a feature adapter function to generate reference images for inversion, providing style templates for input image inversion, called Style Target Path. We utilize the inversion of the input image as the Structural Target path, employing visual prompt images to maintain structural consistency in this path while preserving style information from the style Target path. During the deterministic sampling process, we achieve complete content-style disentanglement through a plug-and-play embedding visual prompt approach. (2) StainPrompt Optimization, where we only optimize the null visual prompt as ``operator'' for dual path inversion, rather than fine-tune pre-trained model. We optimize null visual prompt for structual and style trajectory around pivotal noise on each timestep, ensuring accurate dual-path inversion reconstruction. Extensive evaluations on publicly available multi-domain unpaired staining datasets demonstrate high structural consistency and accurate style transfer results.
Significant biomedical research and clinical care rely on the histopathologic examination of tissue structure using microscopy of stained tissue. Virtual staining (VS) offers a promising alternative with the potential to reduce cost and eliminate the use of toxic reagents. However, the critical challenge of hallucinations limits confidence in its use, necessitating a VS co-pilot to detect these hallucinations. Here, we first formally establish the problem of hallucination detection in VS. Next, we introduce a scalable, post-hoc hallucination detection method that identifies a Neural Hallucination Precursor (NHP) from VS model embeddings for test-time detection. We report extensive validation across diverse and challenging VS settings to demonstrate NHP's effectiveness and robustness. Furthermore, we show that VS models with fewer hallucinations do not necessarily disclose them better, risking a false sense of security when reporting just the former metric. This highlights the need for a reassessment of current VS evaluation practices.
Imaging mass spectrometry (IMS) is a powerful tool for untargeted, highly multiplexed molecular mapping of tissue in biomedical research. IMS offers a means of mapping the spatial distributions of molecular species in biological tissue with unparalleled chemical specificity and sensitivity. However, most IMS platforms are not able to achieve microscopy-level spatial resolution and lack cellular morphological contrast, necessitating subsequent histochemical staining, microscopic imaging and advanced image registration steps to enable molecular distributions to be linked to specific tissue features and cell types. Here, we present a virtual histological staining approach that enhances spatial resolution and digitally introduces cellular morphological contrast into mass spectrometry images of label-free human tissue using a diffusion model. Blind testing on human kidney tissue demonstrated that the virtually stained images of label-free samples closely match their histochemically stained counterparts (with Periodic Acid-Schiff staining), showing high concordance in identifying key renal pathology structures despite utilizing IMS data with 10-fold larger pixel size. Additionally, our approach employs an optimized noise sampling technique during the diffusion model's inference process to reduce variance in the generated images, yielding reliable and repeatable virtual staining. We believe this virtual staining method will significantly expand the applicability of IMS in life sciences and open new avenues for mass spectrometry-based biomedical research.
Virtual staining of tissue offers a powerful tool for transforming label-free microscopy images of unstained tissue into equivalents of histochemically stained samples. This study presents a diffusion model-based super-resolution virtual staining approach utilizing a Brownian bridge process to enhance both the spatial resolution and fidelity of label-free virtual tissue staining, addressing the limitations of traditional deep learning-based methods. Our approach integrates novel sampling techniques into a diffusion model-based image inference process to significantly reduce the variance in the generated virtually stained images, resulting in more stable and accurate outputs. Blindly applied to lower-resolution auto-fluorescence images of label-free human lung tissue samples, the diffusion-based super-resolution virtual staining model consistently outperformed conventional approaches in resolution, structural similarity and perceptual accuracy, successfully achieving a super-resolution factor of 4-5x, increasing the output space-bandwidth product by 16-25-fold compared to the input label-free microscopy images. Diffusion-based super-resolved virtual tissue staining not only improves resolution and image quality but also enhances the reliability of virtual staining without traditional chemical staining, offering significant potential for clinical diagnostics.
The multimodal model has demonstrated promise in histopathology. However, most multimodal models are based on H\&E and genomics, adopting increasingly complex yet black-box designs. In our paper, we propose a novel interpretable multimodal framework named SHAP-CAT, which uses a Shapley-value-based dimension reduction technique for effective multimodal fusion. Starting with two paired modalities -- H\&E and IHC images, we employ virtual staining techniques to enhance limited input data by generating a new clinical-related modality. Lightweight bag-level representations are extracted from image modalities and a Shapley-value-based mechanism is used for dimension reduction. For each dimension of the bag-level representation, attribution values are calculated to indicate how changes in the specific dimensions of the input affect the model output. In this way, we select a few top important dimensions of bag-level representation for each image modality to late fusion. Our experimental results demonstrate that the proposed SHAP-CAT framework incorporating synthetic modalities significantly enhances model performance, yielding a 5\% increase in accuracy for the BCI, an 8\% increase for IHC4BC-ER, and an 11\% increase for the IHC4BC-PR dataset.
Immunohistochemical (IHC) stains play a vital role in a pathologist's analysis of medical images, providing crucial diagnostic information for various diseases. Virtual staining from hematoxylin and eosin (H&E)-stained whole slide images (WSIs) allows the automatic production of other useful IHC stains without the expensive physical staining process. However, current virtual WSI generation methods based on tile-wise processing often suffer from inconsistencies in content, texture, and color at tile boundaries. These inconsistencies lead to artifacts that compromise image quality and potentially hinder accurate clinical assessment and diagnoses. To address this limitation, we propose a novel consistent WSI synthesis network, CC-WSI-Net, that extends GAN models to produce seamless synthetic whole slide images. Our CC-WSI-Net integrates a content- and color-consistency supervisor, ensuring consistency across tiles and facilitating the generation of seamless synthetic WSIs while ensuring Sox10 immunohistochemistry accuracy in melanocyte detection. We validate our method through extensive image-quality analyses, objective detection assessments, and a subjective survey with pathologists. By generating high-quality synthetic WSIs, our method opens doors for advanced virtual staining techniques with broader applications in research and clinical care.
Organ transplantation serves as the primary therapeutic strategy for end-stage organ failures. However, allograft rejection is a common complication of organ transplantation. Histological assessment is essential for the timely detection and diagnosis of transplant rejection and remains the gold standard. Nevertheless, the traditional histochemical staining process is time-consuming, costly, and labor-intensive. Here, we present a panel of virtual staining neural networks for lung and heart transplant biopsies, which digitally convert autofluorescence microscopic images of label-free tissue sections into their brightfield histologically stained counterparts, bypassing the traditional histochemical staining process. Specifically, we virtually generated Hematoxylin and Eosin (H&E), Masson's Trichrome (MT), and Elastic Verhoeff-Van Gieson (EVG) stains for label-free transplant lung tissue, along with H&E and MT stains for label-free transplant heart tissue. Subsequent blind evaluations conducted by three board-certified pathologists have confirmed that the virtual staining networks consistently produce high-quality histology images with high color uniformity, closely resembling their well-stained histochemical counterparts across various tissue features. The use of virtually stained images for the evaluation of transplant biopsies achieved comparable diagnostic outcomes to those obtained via traditional histochemical staining, with a concordance rate of 82.4% for lung samples and 91.7% for heart samples. Moreover, virtual staining models create multiple stains from the same autofluorescence input, eliminating structural mismatches observed between adjacent sections stained in the traditional workflow, while also saving tissue, expert time, and staining costs.
Histological Tertiary Lymphoid Structures (TLSs) are increasingly recognized for their correlation with the efficacy of immunotherapy in various solid tumors. Traditionally, the identification and characterization of TLSs rely on immunohistochemistry (IHC) staining techniques, utilizing markers such as CD20 for B cells. Despite the specificity of IHC, Hematoxylin-Eosin (H&E) staining offers a more accessible and cost-effective choice. Capitalizing on the prevalence of H&E staining slides, we introduce a novel Mask-Guided Adversarial Transfer Learning method designed for virtual pathological staining. This method adeptly captures the nuanced color variations across diverse tissue types under various staining conditions, such as nucleus, red blood cells, positive reaction regions, without explicit label information, and adeptly synthesizes realistic IHC-like virtual staining patches, even replicating the positive reaction. Further, we propose the Virtual IHC Pathology Analysis Network (VIPA-Net), an integrated framework encompassing a Mask-Guided Transfer Module and an H&E-Based Virtual Staining TLS Detection Module. VIPA-Net synergistically harnesses both H\&E staining slides and the synthesized virtual IHC patches to enhance the detection of TLSs within H&E Whole Slide Images (WSIs). We evaluate the network with a comprehensive dataset comprising 1019 annotated slides from The Cancer Genome Atlas (TCGA). Experimental results compellingly illustrate that the VIPA-Net substantially elevates TLS detection accuracy, effectively circumventing the need for actual CD20 staining across the public dataset.
The large volume and variety of imaging data from high-throughput screening (HTS) in the pharmaceutical industry present an excellent resource for training virtual staining models. However, the potential of models trained under one set of experimental conditions to generalize to other conditions remains underexplored. This study systematically investigates whether data from three cell types (lung, ovarian, and breast) and two phenotypes (toxic and non-toxic conditions) commonly found in HTS can effectively train virtual staining models to generalize across three typical HTS distribution shifts: unseen phenotypes, unseen cell types, and the combination of both. Utilizing a dataset of 772,416 paired bright-field, cytoplasm, nuclei, and DNA-damage stain images, we evaluate the generalization capabilities of models across pixel-based, instance-wise, and biological-feature-based levels. Our findings indicate that training virtual nuclei and cytoplasm models on non-toxic condition samples not only generalizes to toxic condition samples but leads to improved performance across all evaluation levels compared to training on toxic condition samples. Generalization to unseen cell types shows variability depending on the cell type; models trained on ovarian or lung cell samples often perform well under other conditions, while those trained on breast cell samples consistently show poor generalization. Generalization to unseen cell types and phenotypes shows good generalization across all levels of evaluation compared to addressing unseen cell types alone. This study represents the first large-scale, data-centric analysis of the generalization capability of virtual staining models trained on diverse HTS datasets, providing valuable strategies for experimental training data generation.
Conventional hematoxylin-eosin (H&E) staining is limited to revealing cell morphology and distribution, whereas immunohistochemical (IHC) staining provides precise and specific visualization of protein activation at the molecular level. Virtual staining technology has emerged as a solution for highly efficient IHC examination, which directly transforms H&E-stained images to IHC-stained images. However, virtual staining is challenged by the insufficient mining of pathological semantics and the spatial misalignment of pathological semantics. To address these issues, we propose the Pathological Semantics-Preserving Learning method for Virtual Staining (PSPStain), which directly incorporates the molecular-level semantic information and enhances semantics interaction despite any spatial inconsistency. Specifically, PSPStain comprises two novel learning strategies: 1) Protein-Aware Learning Strategy (PALS) with Focal Optical Density (FOD) map maintains the coherence of protein expression level, which represents molecular-level semantic information; 2) Prototype-Consistent Learning Strategy (PCLS), which enhances cross-image semantic interaction by prototypical consistency learning. We evaluate PSPStain on two public datasets using five metrics: three clinically relevant metrics and two for image quality. Extensive experiments indicate that PSPStain outperforms current state-of-the-art H&E-to-IHC virtual staining methods and demonstrates a high pathological correlation between the staging of real and virtual stains.
Chemical staining methods are dependable but require extensive time, expensive chemicals, and raise environmental concerns. These challenges highlight the need for alternative solutions like virtual staining, which accelerates the diagnostic process and enhances stain application flexibility. Generative AI technologies are pivotal in addressing these issues. However, the high-stakes nature of healthcare decisions, especially in computational pathology, complicates the adoption of these tools due to their opaque processes. Our work introduces the use of generative AI for virtual staining, aiming to enhance performance, trustworthiness, scalability, and adaptability in computational pathology. The methodology centers on a singular H&E encoder supporting multiple stain decoders. This design focuses on critical regions in the latent space of H&E, enabling precise synthetic stain generation. Our method, tested to generate 8 different stains from a single H&E slide, offers scalability by loading only necessary model components during production. We integrate label-free knowledge in training, using loss functions and regularization to minimize artifacts, thus improving paired/unpaired virtual staining accuracy. To build trust, we use real-time self-inspection with discriminators for each stain type, providing pathologists with confidence heat-maps. Automatic quality checks on new H&E slides ensure conformity to the trained distribution, ensuring accurate synthetic stains. Recognizing pathologists' challenges with new technologies, we have developed an open-source, cloud-based system, that allows easy virtual staining of H&E slides through a browser, addressing hardware/software issues and facilitating real-time user feedback. We also curated a novel dataset of 8 paired H&E/stains related to pediatric Crohn's disease, comprising 480 WSIs to further stimulate computational pathology research.
Virtual staining streamlines traditional staining procedures by digitally generating stained images from unstained or differently stained images. While conventional staining methods involve time-consuming chemical processes, virtual staining offers an efficient and low infrastructure alternative. Leveraging microscopy-based techniques, such as confocal microscopy, researchers can expedite tissue analysis without the need for physical sectioning. However, interpreting grayscale or pseudo-color microscopic images remains a challenge for pathologists and surgeons accustomed to traditional histologically stained images. To fill this gap, various studies explore digitally simulating staining to mimic targeted histological stains. This paper introduces a novel network, In-and-Out Net, specifically designed for virtual staining tasks. Based on Generative Adversarial Networks (GAN), our model efficiently transforms Reflectance Confocal Microscopy (RCM) images into Hematoxylin and Eosin (H&E) stained images. We enhance nuclei contrast in RCM images using aluminum chloride preprocessing for skin tissues. Training the model with virtual H\&E labels featuring two fluorescence channels eliminates the need for image registration and provides pixel-level ground truth. Our contributions include proposing an optimal training strategy, conducting a comparative analysis demonstrating state-of-the-art performance, validating the model through an ablation study, and collecting perfectly matched input and ground truth images without registration. In-and-Out Net showcases promising results, offering a valuable tool for virtual staining tasks and advancing the field of histological image analysis.