Digital pathology has recently been revolutionized by advancements in artificial intelligence, deep learning, and high-performance computing. With its advanced tools, digital pathology can help improve and speed up the diagnostic process, reduce human errors, and streamline the reporting step. In this paper, we report a new large red blood cell (RBC) image dataset and propose a two-stage deep learning framework for RBC image segmentation and classification. The dataset is a highly diverse dataset of more than 100K RBCs containing eight different classes. The dataset, which is considerably larger than any publicly available hematopathology dataset, was labeled independently by two hematopathologists who also manually created masks for RBC cell segmentation. Subsequently, in the proposed framework, first, a U-Net model was trained to achieve automatic RBC image segmentation. Second, an EfficientNetB0 model was trained to classify RBC images into one of the eight classes using a transfer learning approach with a 5X2 cross-validation scheme. An IoU of 98.03% and an average classification accuracy of 96.5% were attained on the test set. Moreover, we have performed experimental comparisons against several prominent CNN models. These comparisons show the superiority of the proposed model with a good balance between performance and computational cost.
The segmentation and tracking of living cells play a vital role within the biomedical domain, particularly in cancer research, drug development, and developmental biology. These are usually tedious and time-consuming tasks that are traditionally done by biomedical experts. Recently, to automatize these processes, deep learning based segmentation and tracking methods have been proposed. These methods require large-scale datasets and their full potential is constrained by the scarcity of annotated data in the biomedical imaging domain. To address this limitation, we propose Biomedical Video Diffusion Model (BVDM), capable of generating realistic-looking synthetic microscopy videos. Trained only on a single real video, BVDM can generate videos of arbitrary length with pixel-level annotations that can be used for training data-hungry models. It is composed of a denoising diffusion probabilistic model (DDPM) generating high-fidelity synthetic cell microscopy images and a flow prediction model (FPM) predicting the non-rigid transformation between consecutive video frames. During inference, initially, the DDPM imposes realistic cell textures on synthetic cell masks which are generated based on real data statistics. The flow prediction model predicts the flow field between consecutive masks and applies that to the DDPM output from the previous time frame to create the next one while keeping temporal consistency. BVDM outperforms state-of-the-art synthetic live cell microscopy video generation models. Furthermore, we demonstrate that a sufficiently large synthetic dataset enhances the performance of cell segmentation and tracking models compared to using a limited amount of available real data.
Hematoxylin and Eosin (H&E) staining is widely regarded as the standard in pathology for diagnosing diseases and tracking tumor recurrence. While H&E staining shows tissue structures, it lacks the ability to reveal specific proteins that are associated with disease severity and treatment response. Immunohistochemical (IHC) stains use antibodies to highlight the expression of these proteins on their respective cell types, improving diagnostic accuracy, and assisting with drug selection for treatment. Despite their value, IHC stains require additional time and resources, limiting their utilization in some clinical settings. Recent advances in deep learning have positioned Image-to-Image (I2I) translation as a computational, cost-effective alternative for IHC. I2I generates high fidelity stain transformations digitally, potentially replacing manual staining in IHC. Diffusion models, the current state of the art in image generation and conditional tasks, are particularly well suited for virtual IHC due to their ability to produce high quality images and resilience to mode collapse. However, these models require extensive and diverse datasets (often millions of samples) to achieve a robust performance, a challenge in virtual staining applications where only thousands of samples are typically available. Inspired by the success of multitask deep learning models in scenarios with limited data, we introduce STAINDIFFUSER, a novel multitask diffusion architecture tailored to virtual staining that achieves convergence with smaller datasets. STAINDIFFUSER simultaneously trains two diffusion processes: (a) generating cell specific IHC stains from H&E images and (b) performing H&E based cell segmentation, utilizing coarse segmentation labels exclusively during training. STAINDIFFUSER generates high-quality virtual stains for two markers, outperforming over twenty I2I baselines.
Imaging mass cytometry (IMC) is a relatively new technique for imaging biological tissue at subcellular resolution. In recent years, learning-based segmentation methods have enabled precise quantification of cell type and morphology, but typically rely on large datasets with fully annotated ground truth (GT) labels. This paper explores the effects of imperfect labels on learning-based segmentation models and evaluates the generalisability of these models to different tissue types. Our results show that removing 50% of cell annotations from GT masks only reduces the dice similarity coefficient (DSC) score to 0.874 (from 0.889 achieved by a model trained on fully annotated GT masks). This implies that annotation time can in fact be reduced by at least half without detrimentally affecting performance. Furthermore, training our single-tissue model on imperfect labels only decreases DSC by 0.031 on an unseen tissue type compared to its multi-tissue counterpart, with negligible qualitative differences in segmentation. Additionally, bootstrapping the worst-performing model (with 5% of cell annotations) a total of ten times improves its original DSC score of 0.720 to 0.829. These findings imply that less time and work can be put into the process of producing comparable segmentation models; this includes eliminating the need for multiple IMC tissue types during training, whilst also providing the potential for models with very few labels to improve on themselves. Source code is available on GitHub: https://github.com/kimberley/ISBI2024.
Artificially generated induced pluripotent stem cells (iPSCs) from somatic cells play an important role for disease modeling and drug screening of neurodegenerative diseases. Astrocytes differentiated from iPSCs are important targets to investigate neuronal metabolism. The astrocyte differentiation progress can be monitored through the variations of morphology observed from microscopy images at different differentiation stages, then determined by molecular biology techniques upon maturation. However, the astrocytes usually ``perfectly'' blend into the background and some of them are covered by interference information (i.e., dead cells, media sediments, and cell debris), which makes astrocytes difficult to observe. Due to the lack of annotated datasets, the existing state-of-the-art deep learning approaches cannot be used to address this issue. In this paper, we introduce a new task named astrocyte segmentation with a novel dataset, called IAI704, which contains 704 images and their corresponding pixel-level annotation masks. Moreover, a novel frequency domain denoising network, named FDNet, is proposed for astrocyte segmentation. In detail, our FDNet consists of a contextual information fusion module (CIF), an attention block (AB), and a Fourier transform block (FTB). CIF and AB fuse multi-scale feature embeddings to localize the astrocytes. FTB transforms feature embeddings into the frequency domain and conducts a high-pass filter to eliminate interference information. Experimental results demonstrate the superiority of our proposed FDNet over the state-of-the-art substitutes in astrocyte segmentation, shedding insights for iPSC differentiation progress prediction.
Background: Cell segmentation in bright-field histological slides is a crucial topic in medical image analysis. Having access to accurate segmentation allows researchers to examine the relationship between cellular morphology and clinical observations. Unfortunately, most segmentation methods known today are limited to nuclei and cannot segmentate the cytoplasm. Material & Methods: We present a new network architecture Cyto R-CNN that is able to accurately segment whole cells (with both the nucleus and the cytoplasm) in bright-field images. We also present a new dataset CytoNuke, consisting of multiple thousand manual annotations of head and neck squamous cell carcinoma cells. Utilizing this dataset, we compared the performance of Cyto R-CNN to other popular cell segmentation algorithms, including QuPath's built-in algorithm, StarDist and Cellpose. To evaluate segmentation performance, we calculated AP50, AP75 and measured 17 morphological and staining-related features for all detected cells. We compared these measurements to the gold standard of manual segmentation using the Kolmogorov-Smirnov test. Results: Cyto R-CNN achieved an AP50 of 58.65% and an AP75 of 11.56% in whole-cell segmentation, outperforming all other methods (QuPath $19.46/0.91\%$; StarDist $45.33/2.32\%$; Cellpose $31.85/5.61\%$). Cell features derived from Cyto R-CNN showed the best agreement to the gold standard ($\bar{D} = 0.15$) outperforming QuPath ($\bar{D} = 0.22$), StarDist ($\bar{D} = 0.25$) and Cellpose ($\bar{D} = 0.23$). Conclusion: Our newly proposed Cyto R-CNN architecture outperforms current algorithms in whole-cell segmentation while providing more reliable cell measurements than any other model. This could improve digital pathology workflows, potentially leading to improved diagnosis. Moreover, our published dataset can be used to develop further models in the future.
Convolutional Neural Networks (CNNs) and Transformers have been the most popular architectures for biomedical image segmentation, but both of them have limited ability to handle long-range dependencies because of inherent locality or computational complexity. To address this challenge, we introduce U-Mamba, a general-purpose network for biomedical image segmentation. Inspired by the State Space Sequence Models (SSMs), a new family of deep sequence models known for their strong capability in handling long sequences, we design a hybrid CNN-SSM block that integrates the local feature extraction power of convolutional layers with the abilities of SSMs for capturing the long-range dependency. Moreover, U-Mamba enjoys a self-configuring mechanism, allowing it to automatically adapt to various datasets without manual intervention. We conduct extensive experiments on four diverse tasks, including the 3D abdominal organ segmentation in CT and MR images, instrument segmentation in endoscopy images, and cell segmentation in microscopy images. The results reveal that U-Mamba outperforms state-of-the-art CNN-based and Transformer-based segmentation networks across all tasks. This opens new avenues for efficient long-range dependency modeling in biomedical image analysis. The code, models, and data are publicly available at https://wanglab.ai/u-mamba.html.
Morphological profiling is a valuable tool in phenotypic drug discovery. The advent of high-throughput automated imaging has enabled the capturing of a wide range of morphological features of cells or organisms in response to perturbations at the single-cell resolution. Concurrently, significant advances in machine learning and deep learning, especially in computer vision, have led to substantial improvements in analyzing large-scale high-content images at high-throughput. These efforts have facilitated understanding of compound mechanism-of-action (MOA), drug repurposing, characterization of cell morphodynamics under perturbation, and ultimately contributing to the development of novel therapeutics. In this review, we provide a comprehensive overview of the recent advances in the field of morphological profiling. We summarize the image profiling analysis workflow, survey a broad spectrum of analysis strategies encompassing feature engineering- and deep learning-based approaches, and introduce publicly available benchmark datasets. We place a particular emphasis on the application of deep learning in this pipeline, covering cell segmentation, image representation learning, and multimodal learning. Additionally, we illuminate the application of morphological profiling in phenotypic drug discovery and highlight potential challenges and opportunities in this field.
In recent years, several unsupervised cell segmentation methods have been presented, trying to omit the requirement of laborious pixel-level annotations for the training of a cell segmentation model. Most if not all of these methods handle the instance segmentation task by focusing on the detection of different cell instances ignoring their type. While such models prove adequate for certain tasks, like cell counting, other applications require the identification of each cell's type. In this paper, we present CellMixer, an innovative annotation-free approach for the semantic segmentation of heterogeneous cell populations. Our augmentation-based method enables the training of a segmentation model from image-level labels of homogeneous cell populations. Our results show that CellMixer can achieve competitive segmentation performance across multiple cell types and imaging modalities, demonstrating the method's scalability and potential for broader applications in medical imaging, cellular biology, and diagnostics.
Cell segmentation in histopathological images plays a crucial role in understanding, diagnosing, and treating many diseases. However, data annotation for this is expensive since there can be a large number of cells per image, and expert pathologists are needed for labelling images. Instead, our paper focuses on using weak supervision -- annotation from related tasks -- to induce a segmenter. Recent foundation models, such as Segment Anything (SAM), can use prompts to leverage additional supervision during inference. SAM has performed remarkably well in natural image segmentation tasks; however, its applicability to cell segmentation has not been explored. In response, we investigate guiding the prompting procedure in SAM for weakly supervised cell segmentation when only bounding box supervision is available. We develop two workflows: (1) an object detector's output as a test-time prompt to SAM (D-SAM), and (2) SAM as pseudo mask generator over training data to train a standalone segmentation model (SAM-S). On finding that both workflows have some complementary strengths, we develop an integer programming-based approach to reconcile the two sets of segmentation masks, achieving yet higher performance. We experiment on three publicly available cell segmentation datasets namely, ConSep, MoNuSeg, and TNBC, and find that all SAM-based solutions hugely outperform existing weakly supervised image segmentation models, obtaining 9-15 pt Dice gains.
Recent advances in imaging and high-performance computing have made it possible to image the entire human brain at the cellular level. This is the basis to study the multi-scale architecture of the brain regarding its subdivision into brain areas and nuclei, cortical layers, columns, and cell clusters down to single cell morphology Methods for brain mapping and cell segmentation exploit such images to enable rapid and automated analysis of cytoarchitecture and cell distribution in complete series of histological sections. However, the presence of inevitable processing artifacts in the image data caused by missing sections, tears in the tissue, or staining variations remains the primary reason for gaps in the resulting image data. To this end we aim to provide a model that can fill in missing information in a reliable way, following the true cell distribution at different scales. Inspired by the recent success in image generation, we propose a denoising diffusion probabilistic model (DDPM), trained on light-microscopic scans of cell-body stained sections. We extend this model with the RePaint method to impute missing or replace corrupted image data. We show that our trained DDPM is able to generate highly realistic image information for this purpose, generating plausible cell statistics and cytoarchitectonic patterns. We validate its outputs using two established downstream task models trained on the same data.
Cells are the fundamental unit of biological organization, and identifying them in imaging data - cell segmentation - is a critical task for various cellular imaging experiments. While deep learning methods have led to substantial progress on this problem, models that have seen wide use are specialist models that work well for specific domains. Methods that have learned the general notion of "what is a cell" and can identify them across different domains of cellular imaging data have proven elusive. In this work, we present CellSAM, a foundation model for cell segmentation that generalizes across diverse cellular imaging data. CellSAM builds on top of the Segment Anything Model (SAM) by developing a prompt engineering approach to mask generation. We train an object detector, CellFinder, to automatically detect cells and prompt SAM to generate segmentations. We show that this approach allows a single model to achieve state-of-the-art performance for segmenting images of mammalian cells (in tissues and cell culture), yeast, and bacteria collected with various imaging modalities. To enable accessibility, we integrate CellSAM into DeepCell Label to further accelerate human-in-the-loop labeling strategies for cellular imaging data. A deployed version of CellSAM is available at https://label-dev.deepcell.org/.
Segmentation, or the outlining of objects within images, is a critical step in the measurement and analysis of cells within microscopy images. While improvements continue to be made in tools that rely on classical methods for segmentation, deep learning-based tools increasingly dominate advances in the technology. Specialist models such as Cellpose continue to improve in accuracy and user-friendliness, and segmentation challenges such as the Multi-Modality Cell Segmentation Challenge continue to push innovation in accuracy across widely-varying test data as well as efficiency and usability. Increased attention on documentation, sharing, and evaluation standards are leading to increased user-friendliness and acceleration towards the goal of a truly universal method.
Extracting long tracks and lineages from videomicroscopy requires an extremely low error rate, which is challenging on complex datasets of dense or deforming cells. Leveraging temporal context is key to overcoming this challenge. We propose DistNet2D, a new deep neural network (DNN) architecture for 2D cell segmentation and tracking that leverages both mid- and long-term temporal information. DistNet2D considers seven frames at the input and uses a post-processing procedure that exploits information from the entire video to correct segmentation errors. DistNet2D outperforms two recent methods on two experimental datasets, one containing densely packed bacterial cells and the other containing eukaryotic cells. It is integrated into an ImageJ-based graphical user interface for 2D data visualization, curation, and training. Finally, we demonstrate the performance of DistNet2D on correlating the size and shape of cells with their transport properties over large statistics, for both bacterial and eukaryotic cells.
Cell segmentation for multi-modal microscopy images remains a challenge due to the complex textures, patterns, and cell shapes in these images. To tackle the problem, we first develop an automatic cell classification pipeline to label the microscopy images based on their low-level image characteristics, and then train a classification model based on the category labels. Afterward, we train a separate segmentation model for each category using the images in the corresponding category. Besides, we further deploy two types of segmentation models to segment cells with roundish and irregular shapes respectively. Moreover, an efficient and powerful backbone model is utilized to enhance the efficiency of our segmentation model. Evaluated on the Tuning Set of NeurIPS 2022 Cell Segmentation Challenge, our method achieves an F1-score of 0.8795 and the running time for all cases is within the time tolerance.
While pre-training on object detection tasks, such as Common Objects in Contexts (COCO) [1], could significantly boost the performance of cell segmentation, it still consumes on massive fine-annotated cell images [2] with bounding boxes, masks, and cell types for every cell in every image, to fine-tune the pre-trained model. To lower the cost of annotation, this work considers the problem of pre-training DNN models for few-shot cell segmentation, where massive unlabeled cell images are available but only a small proportion is annotated. Hereby, we propose Cross-domain Unsupervised Pre-training, namely CUPre, transferring the capability of object detection and instance segmentation for common visual objects (learned from COCO) to the visual domain of cells using unlabeled images. Given a standard COCO pre-trained network with backbone, neck, and head modules, CUPre adopts an alternate multi-task pre-training (AMT2) procedure with two sub-tasks -- in every iteration of pre-training, AMT2 first trains the backbone with cell images from multiple cell datasets via unsupervised momentum contrastive learning (MoCo) [3], and then trains the whole model with vanilla COCO datasets via instance segmentation. After pre-training, CUPre fine-tunes the whole model on the cell segmentation task using a few annotated images. We carry out extensive experiments to evaluate CUPre using LIVECell [2] and BBBC038 [4] datasets in few-shot instance segmentation settings. The experiment shows that CUPre can outperform existing pre-training methods, achieving the highest average precision (AP) for few-shot cell segmentation and detection.
We propose DiRL, a Diversity-inducing Representation Learning technique for histopathology imaging. Self-supervised learning techniques, such as contrastive and non-contrastive approaches, have been shown to learn rich and effective representations of digitized tissue samples with limited pathologist supervision. Our analysis of vanilla SSL-pretrained models' attention distribution reveals an insightful observation: sparsity in attention, i.e, models tends to localize most of their attention to some prominent patterns in the image. Although attention sparsity can be beneficial in natural images due to these prominent patterns being the object of interest itself, this can be sub-optimal in digital pathology; this is because, unlike natural images, digital pathology scans are not object-centric, but rather a complex phenotype of various spatially intermixed biological components. Inadequate diversification of attention in these complex images could result in crucial information loss. To address this, we leverage cell segmentation to densely extract multiple histopathology-specific representations, and then propose a prior-guided dense pretext task for SSL, designed to match the multiple corresponding representations between the views. Through this, the model learns to attend to various components more closely and evenly, thus inducing adequate diversification in attention for capturing context rich representations. Through quantitative and qualitative analysis on multiple tasks across cancer types, we demonstrate the efficacy of our method and observe that the attention is more globally distributed.
To date, most instance segmentation approaches are based on supervised learning that requires a considerable amount of annotated object contours as training ground truth. Here, we propose a framework that searches for the target object based on a shape prior. The shape prior model is learned with a variational autoencoder that requires only a very limited amount of training data: In our experiments, a few dozens of object shape patches from the target dataset, as well as purely synthetic shapes, were sufficient to achieve results en par with supervised methods with full access to training data on two out of three cell segmentation datasets. Our method with a synthetic shape prior was superior to pre-trained supervised models with access to limited domain-specific training data on all three datasets. Since the learning of prior models requires shape patches, whether real or synthetic data, we call this framework semi-supervised learning.
Image segmentation plays an essential role in nuclei image analysis. Recently, the segment anything model has made a significant breakthrough in such tasks. However, the current model exists two major issues for cell segmentation: (1) the image encoder of the segment anything model involves a large number of parameters. Retraining or even fine-tuning the model still requires expensive computational resources. (2) in point prompt mode, points are sampled from the center of the ground truth and more than one set of points is expected to achieve reliable performance, which is not efficient for practical applications. In this paper, a single-point prompt network is proposed for nuclei image segmentation, called SPPNet. We replace the original image encoder with a lightweight vision transformer. Also, an effective convolutional block is added in parallel to extract the low-level semantic information from the image and compensate for the performance degradation due to the small image encoder. We propose a new point-sampling method based on the Gaussian kernel. The proposed model is evaluated on the MoNuSeg-2018 dataset. The result demonstrated that SPPNet outperforms existing U-shape architectures and shows faster convergence in training. Compared to the segment anything model, SPPNet shows roughly 20 times faster inference, with 1/70 parameters and computational cost. Particularly, only one set of points is required in both the training and inference phases, which is more reasonable for clinical applications. The code for our work and more technical details can be found at https://github.com/xq141839/SPPNet.
The success of supervised deep learning models on cell recognition tasks relies on detailed annotations. Many previous works have managed to reduce the dependency on labels. However, considering the large number of cells contained in a patch, costly and inefficient labeling is still inevitable. To this end, we explored label-free methods for cell recognition. Prior self-activation maps (PSM) are proposed to generate pseudo masks as training targets. To be specific, an activation network is trained with self-supervised learning. The gradient information in the shallow layers of the network is aggregated to generate prior self-activation maps. Afterward, a semantic clustering module is then introduced as a pipeline to transform PSMs to pixel-level semantic pseudo masks for downstream tasks. We evaluated our method on two histological datasets: MoNuSeg (cell segmentation) and BCData (multi-class cell detection). Compared with other fully-supervised and weakly-supervised methods, our method can achieve competitive performance without any manual annotations. Our simple but effective framework can also achieve multi-class cell detection which can not be done by existing unsupervised methods. The results show the potential of PSMs that might inspire other research to deal with the hunger for labels in medical area.