Microscopy Image Analysis

Cell viability assessment is a core requirement in cell culture systems, with critical applications in biopharmaceutical manufacturing and drug development. Conventionally, it is measured by adding membrane-impermeable dyes to a sample (a process called staining), which allows compromised cell membranes to be distinguished from intact ones. However, staining has several limitations: (a) chemical agents can perturb normal cellular processes of the cells being measured, (b) it is often ambiguous to assign viability to individual cells whose membrane integrity is only partially compromised. (c) photobleaching can undermine measurement accuracy over time when using fluorescent stains, and (d) staining cannot be performed in situ or in real time. Here, we show that (1) stained cells captured under brightfield imaging contain sufficient information to distinguish live and dead cells, and (2) cells captured under unstained brightfield imaging exhibit similar image features to their stained counterparts, enabling models trained on stained cells to generalize to unstained ones. We then report the development and validation of ViabiLens, an AI-assisted software for label-free cell viability analysis. The ViabiLens combines a cell detection model for localizing individual cells with a convolutional neural network (CNN) classifier for live/dead prediction, paired with an interactive UMAP-based viewer for visualizing and exploring individual cells across the sample. Evaluated on Chinese Hamster Ovary (CHO) cells spanning a wide range of viability conditions, ViabiLens achieves a mean absolute error of 2.68% on unstained samples against fluorescence-based reference measurements. We also release a benchmark dataset for label-free cell viability analysis to facilitate future research, available at https://amirrezavazifeh.github.io/ViabiLens-Project-Page/.

Volume electron microscopy (vEM) has emerged as an essential sensing technique in biomedical research, allowing the three-dimensional imaging of biological cells and tissues at nanometer-scale resolution. The ability to generate extensive datasets has reached the limitations of downstream analysis processes, which depend significantly on the intervention of human experts for preprocessing and annotation. We propose an efficient and reliable patch-based retrieval framework based on self-supervised learning of local image descriptors to locate self-similar structures in vEM datasets. Given a few manual annotations of a given cellular structure, our method can retrieve similar structures across the EM volume. Our framework is interactive, allowing the human expert to refine the search queries and retrieve relevant image patches quickly and using little labeled data. Experiments on real-world vEM images of biological tissues demonstrate that our framework can reliably identify relevant cellular structures, generalize across different organelles and acquisition modalities, and substantially reduce the search space for downstream analysis.

Process control of advanced semiconductor nodes is not only pushing the limits of metrology equipment requirements in terms of resolution and throughput but also in terms of the richness of data to be extracted to enable engineers to finetune the process steps for increased yield. The move towards 3D structures requires extraction of critical dimension parameters from structures which can vary largely from layer to layer. For in-line process control, the necessary automation forces the development of layer and equipment-specific dedicated image processing algorithms. Similarly, with the increase in stochastic defects in the EUV era, detection of defects at the nm scale requires the identification of features captured in low resolution to meet the throughput requirements of HVM fabs, which can again lead to custom algorithm development. With the emergence of ML-based image processing methods, this process of algorithm development for both cases can be accelerated. In this work, we provide the general framework under which the images obtained from high-speed scanning probe microscopy-based systems can be used to train a network for either feature detection for parameter extraction or defect identification.

Background and Objectives: Fluorescence-labeled cellular arbors provide readouts of neuronal and microglial morphology, but fine and weakly labeled processes are prone to fragmentation and false connections that bias skeleton-based measurements. We present MorphoBranch, a fine-structure-preserving, human-reviewable workbench for morphometry of branched cellular structures. Methods: MorphoBranch combines a deterministic Morphometry Engine with an LLM-assisted Refinement Engine. The Mor- phometry Engine implements an image-to-graph workflow integrating multiscale structural evidence extraction, hysteresis segmen- tation, evidence-constrained skeleton refinement, and graph-based morphometry. The Refinement Engine maps natural-language requests to registered actions for parameter adjustment, preview execution, metric reporting, and unsupported-request handling, while image processing and quantitative computation remain deterministic and reviewable. Results: MorphoBranch was evaluated on two public neuronal axon datasets, AxonMIP and AxonStack, and the in-house Cell- Morph dataset of microglial fluorescence images. It achieved the highest Skeleton F1 and clDice and the lowest length-estimation error among the evaluated methods on all three datasets, while also achieving the highest Dice and IoU on AxonMIP and Axon- Stack. Across 150 natural-language tasks, the Refinement Engine achieved a 94.0% end-to-end success rate. Conclusions: These results demonstrate that MorphoBranch provides a reproducible, human-reviewable workflow for mor- phometric analysis of branched cellular structures. It supports fine-structure-preserving quantification across neuronal axon and microglial fluorescence images while maintaining inspectable and reproducible analysis workflows.

Accurate segmentation of axons in 3D microscopy data is important for analyzing white-matter organization, but dense ground truth labels are expensive to obtain. Existing supervised axon segmentation methods rely on target-domain annotations and can be brittle when tissue type, species, modality, or acquisition conditions change. We present AxonSynth, a domain-randomized synthetic-data framework for training 3D axon segmentation models without manually annotated real training volumes. AxonSynth generates dense synthetic axon labels with orientation priors that reflect realistic fiber configurations and renders them with randomized density, contrast, bias fields, blur, and noise. A three-class 3D U-Net is trained to predict background, axon sheath and intra-axonal space. We evaluate zero-shot transfer on 10 held-out light-sheet microscopy (LSM) patches from macaque and human brain samples labeled with one of three axonal markers, comparing against calibrated thresholding and Frangi filtering using overlap, corrected detection, false-positive, and topology metrics. On macaque samples, AxonSynth achieved the best corrected Dice and corrected precision (0.826 and 0.851), compared with 0.765 and 0.754 for thresholding and 0.685 and 0.762 for Frangi. On human samples, corrected Dice was comparable to thresholding (0.857 vs. 0.868), while component-count error decreased from 22,504 to 3,377. Across all held-out patches, AxonSynth reduced component-count error in 10/10 patches and Euler-characteristic error in 8/10. These results show that synthetic-label domain randomization can reduce dependence on manual axon annotation while supporting synthetic-to-real 3D segmentation.

Scanning electron microscopy (SEM) is routinely used to characterize the microstructural changes caused by hydrogen embrittlement (HE) in structural steels. Machine learning can automate this characterization, but models are often evaluated using image-level splits. When several images come from the same specimen region, such splits leak information between the training and test sets. Here, we propose a region-held-out protocol for classifying as-received (AR) and hydrogen-charged (H2) SEM micrographs of 316L stainless steel, based on Leave-One-Region-Out (LORO) cross-validation over 14 spatial regions (8 AR, 6 H2; 31 images). We compared six feature-classifier combinations built on local binary patterns (LBP), grey-level co-occurrence matrices (GLCM), self-supervised convolutional embeddings pretrained on 143 unlabeled SEM images, and a convolutional neural network (CNN). The simplest texture approach, LBP with a support vector machine (LBP+SVM), performed best, achieving a balanced accuracy of 0.79, H2 recall of 0.69, and H2 precision of 0.82, outperforming every deep-learning and combined-feature model. A group-level permutation test (500 permutations sampled from the 3,003 possible region-to-label assignments) yielded p = 0.008, indicating that the result cannot be explained by a chance alignment of the region structure. Grad-CAM maps from a CNN trained on the full dataset tended to concentrate on localized surface and grain-boundary features, where hydrogen-induced morphological changes are known to occur. Under a leakage-safe, statistically validated protocol, texture descriptors recover a hydrogen-charging signature from SEM micrographs even with few samples, and the same protocol can be extended to larger HE detection studies in other alloy systems.

A lack of high-throughput phenotyping solutions for root traits in field-grown crops has severely constrained understanding and improvement of below-ground traits and processes. Minirhizotrons are the standard non-destructive root-phenotyping method in field environments. Computer vision solutions are needed to allow automated trait estimation at scale, but training data is scarce and human annotations are often inaccessible because they reside in proprietary software that only exports per-image scalar totals of root length and surface area. Nevertheless, large numeric archives of these root traits already exist. RootQuant showed that the traits can be predicted directly from the whole image by regression, thus removing manually traced masks from the pipeline; RootQuantV2 takes that idea further by replacing RootQuant's CNN backbone with a self-supervised ViT. We adapt a frozen DINOv3 ViT-L/16 with a hybrid parameter-efficient scheme. Training only 11.9M parameters (3.78% of the model), RootQuantV2 achieves length and area $R^2$ of 0.950 and 0.930, respectively, while lowering length/area RMSE by 24.3%/20.7% over RootQuant. RootQuantV2 thus repurposes legacy numeric archives for high-throughput, automated root trait estimation.

Mitotic figure (MF) analysis supports tumor grading and prognostic assessment, but automated models remain sensitive to differences in tissue type and image acquisition. We present MiTHras, a task-specific pretraining framework that combines pseudo-label-guided image- and token-level contrastive learning with masked reconstruction. We construct TCGA-MF-Pseudo, a corpus of 1.8 million cell-centered images from 14 TCGA cohorts spanning 11 organ sites. Comprehensive evaluation on MF classification, detection, count-based survival prediction, and subtype classification demonstrates the efficacy of MiTHras. It achieves the highest mean F1 on all three MF classification benchmarks and both subtype benchmarks. MiTHras also outperforms general-purpose and pathology foundation encoders by a larger margin under frozen-encoder linear probing than under full fine-tuning. Although detection gains are modest due to a shared candidate-detection stage, ablations confirm that token-level supervision improves typical-versus-atypical classification and linear probing. These findings establish that MiTHras yields robust, transferable representations for automated mitotic activity assessment.

Multimodal large language models (MLLMs) are increasingly explored as interfaces for scientific image analysis, where a visual question-answering (VQA) response may be paired with a spatial output that guides a downstream stage. A supervisor reads the language answer, while a downstream workflow such as segmentation or region review consumes the point-set output. We call this transition from inspecting the answer to relying on its point action the language-to-action hand-off. A silent failure occurs when the answer is correct while the paired action misses annotated objects needed downstream, so answer-based oversight clears a region whose action is unreliable. We introduce Spatial Action Review, a visual analytics dashboard for auditing this failure mode in electron microscopy (EM) mitochondria analysis. It links paired answer-action records through an answer-action ledger, a task-by-dataset risk map, and an image-region audit view, connecting aggregate patterns to image evidence while an adjustable action-reliability gate supports re-audit. The review ends in a human-AI hand-off, where a supervisor records whether the action is accepted, escalated, held under a stricter gate, or flagged for model revision. Across 541 image regions from an EM-adapted Qwen3-VL case-study run, point actions fail the gate in 54.4% of records with a correct VQA response, and 27.4% of all records are silent failures. A correct answer is associated with only a 5.8-percentage-point higher probability of a reliable action, with a bootstrap interval spanning zero; the point-biserial correlation between answer correctness and object coverage is 0.061. This weak coupling persists across five model conditions on 753 matched image regions. Spatial Action Review makes answer-action mismatches visible and ties them to image evidence and a recorded decision before MLLM outputs enter autonomous scientific workflows.

We train neural networks on synthesized frames to approach the optimum Bayes estimator for dense emitter localization. The result justifies the future work on training neural networks to achieve high-throughput large-FOV super spatiotemporal resolution SMLM.

Choosing a deep learning architecture for label-free single-cell classification remains an open question, with microscopy benchmarks reporting conflicting conclusions about CNNs versus transformers. We present a controlled benchmark on LIVECell phase-contrast microscopy data using source-image-disjoint train/validation/test splits to prevent parent-image leakage and matched optimisation, augmentation, and evaluation protocols across EfficientNet, Vision Transformer (ViT), and EVA-02 models. This allows the effects of architecture, pretraining, fine-tuning, tokenisation, and distillation to be disentangled. We find that the previously reported CNN advantage is largely explained by pretraining rather than architecture: the smallest pretrained model outperforms the strongest model trained from scratch despite far fewer parameters. Pretraining improves macro-F1 by 3-4 points, while the gap between the best pretrained CNN and transformer is below 0.5 points. Architectural choices nevertheless matter: ViT-S/8 outperforms ViT-S/16 and matches the four-times-larger ViT-B/16 at a quarter of the parameters, showing that finer tokenisation benefits small cell crops. Conversely, layer-wise learning-rate decay, central to the EVA-02 fine-tuning recipe, degrades performance, highlighting that transfer heuristics from natural-image recognition may not generalise to microscopy. Finally, knowledge distillation substantially improves the deployment frontier: compact EfficientNet-B0 students distilled from teacher councils outperform every individually trained backbone, including the EfficientNet-B5 and EVA-02 teachers. Overall, our results show that rigorous control of pretraining and evaluation is essential for interpreting biomedical architecture benchmarks, while distillation may be a more effective route to practical single-cell classification than architecture choice alone.

Recent advances in high-resolution fluorescence and electron microscopy have enabled nanoscale imaging across increasingly large brain volumes, but the resulting terabyte- to petabyte-scale datasets make complete neuronal reconstruction prohibitively expensive in computation, data movement, and manual proofreading. Here, we present SkNeXt, a topology-first framework for scalable neuronal reconstruction from large volumetric microscopy datasets. Instead of densely processing entire image volumes, SkNeXt first converts neuronal morphology into compact SWC skeletons that preserve long-range connectivity. Proofreading is therefore focused on sparse neuronal trees, allowing branch, continuity, and connectivity errors to be corrected before high-resolution reconstruction. The corrected skeletons then serve as persistent structural priors for recovering detailed morphology while preserving neuronal identity and topology. Crucially, SkNeXt also uses neuronal skeletons as spatial indices for selective data access, retrieving high-resolution image regions only along reconstructed trajectories and bypassing most background and signal-free volumes. This substantially reduces I/O and computational overhead, allowing reconstruction cost to scale with neuronal morphology rather than total dataset size. Using SkNeXt, we reconstructed neurons from a petabyte-scale super-resolution fluorescence dataset of the mouse brain on a single GPU within one week, without requiring exhaustive dense inference across the complete imaging volume.

Organoids are three-dimensional tissue models whose morphology provides important insights into tumor development, disease progression, and drug testing. Extracting these morphological features relies heavily on manual segmentation, which is time-consuming and labor-intensive. Furthermore, performing quantitative statistical analysis typically requires custom coding skills and a mathematical background, presenting a major barrier for experimental biologists. To address these challenges, we introduce MorphoOrgaAgent, a multi-agent framework that achieves zero-shot organoid segmentation, automated data analysis, and report generation based on natural language input. The framework consists mainly of three core components: a TaskUnderstandingAgent that identifies requested measurements and visualization types; a hybrid segmentation module that combines Cellpose-derived geometric prompts with text prompts to guide SAM3 for zero-shot organoid instance segmentation; and a ReportAgent that computes quantitative metrics and compiles them alongside generated visualizations into a structured report. We further introduce MorphoOrgaVQA, a benchmark designed for quantitative evaluation of agent systems in organoid morphology analysis. Experimental results demonstrate that MorphoOrgaAgent handles both explicit and descriptive user requests, produces measurements closely matching ground truth, and generates complete analysis reports without requiring manual programming. The complete source code and MorphoOrgaVQA benchmark are publicly available at https://github.com/peng-lab/MorphoOrgaAgent.

Microscopic image analysis has long been recognized as a promising approach for monitoring activated sludge. In recent years, deep learning-based image analysis has been increasingly adopted in this field because of its high performance. However, previous studies on microscopic image analysis of activated sludge have rarely explored transformer-based models or self-supervised foundation models and have instead relied on CNNs and supervised ImageNet pretraining. In addition, previous studies often downsampled image sizes, but the effects of downsampling have not been sufficiently investigated, and the relationship between downsampling strategies and image analysis performance remains unclear. Furthermore, no study has quantitatively compared deep learning performance with quantitative image analysis (QIA), which was widely used before the emergence of deep learning. In this study, to examine how model architecture and learning strategies affect performance in microscopic image analysis of activated sludge and to quantitatively determine whether deep learning outperforms QIA, we prepared three types of activated sludge samples, classified their microscopic images, and evaluated classification accuracy. Our results showed that transformer-based architectures and alternative pretraining methods were effective in terms of classification accuracy. Our downsampling analysis showed that using overly small images reduced accuracy, but increasing image size beyond a certain point did not improve it further. In addition, the analysis indicated that, to achieve high classification accuracy, maintaining the field of view was a more effective downsampling strategy than maintaining resolution. Finally, our comparison between deep learning and QIA showed that deep learning outperformed QIA in terms of accuracy.

Automating filament tracing in Cryo-Electron Microscopy (Cryo-EM) is essential for 3D helical reconstruction but challenged by intersecting topologies and extremely low Signal-to-Noise Ratios ($\text{SNR} = σ_s^2/σ_n^2$ < 0.1 or -10 dB). Existing paradigms fail: pixel-wise segmenters suffer from severe topological fracturing, box-based detectors face ghost center drift, sequential trackers derail due to error accumulation, and traditional active contours collapse under artificial closed-curve constraints. To resolve these bottlenecks, we present FPicker, the first topology-guided framework reconciling these incompatibilities. It unifies perception via a center-endpoint representation and an open-curve evolution module to explicitly model non-cyclic connectivity. On simulated benchmarks, FPicker outperforms top baselines by over $40\%$ relative gain in mean spatio-angular precision (mSAP) and reduces topological gap rates by over $60\%$ under extreme noise ($-20\text{ dB}$). By learning intrinsic physical geometry rather than local texture, FPicker demonstrates strong potential as a resilient geometric backbone. Its zero-shot performance on the real-world EMPIAR dataset exhibits robust topological resistance, achieving a state-of-the-art 82.9% mSAP upon fine-tuning. Our results also suggest modeling physical priors is a highly robust path toward bridging the sim-to-real gap in signal-starved scientific imaging. The code is publicly available at: https://github.com/tomzhaosky/FPicker.

Morphology of the sub-basal nerve plexus (SNP) reflects peripheral nerve health, and corneal confocal microscopy (CCM) provides an important means for in vivo, real-time, non-invasive observation of the SNP. However, mainstream CCM devices offer a limited field of view per frame, whereas the SNP is spatially non-uniform; discrete image sampling is therefore sensitive to sampling location and frame selection, which limits the reproducibility and clinical adoption of CCM as a quantitative assessment tool. Wide-field stitching can reconstruct larger SNP mosaics by integrating sequentially acquired CCM images, but existing methods largely rely on offline post-processing, additional hardware, or specific acquisition protocols, and lack open-source real-time solutions for conventional CCM video streams. This paper presents CRISP (Corneal confocal microscopy Real-time Image Stitching Pipeline), an open-source real-time SNP wide-field stitching framework for conventional CCM examination video streams. CRISP excludes defocused and discontinuous segments via focus-aware gating, propagates poses through local pairwise registration, and maintains non-redundant spatial coverage with a sparse anchor map; when local temporal continuity is interrupted, the system completes relocalization and subgraph merging through global appearance retrieval followed by geometric verification. The framework prioritizes low-latency coverage feedback during examination while outputting accepted frames, poses, and anchor information to initialize offline fine stitching. To our knowledge, CRISP is the first open-source real-time SNP wide-field stitching framework released for conventional CCM video streams. By lowering the barrier to adoption and reproduction of wide-field stitching, CRISP may help move SNP wide-field imaging from a research tool into routine clinical examination workflows.

Segmenting a new biomedical dataset usually means a domain-specific model trained on substantial annotation, or a foundation model steered at inference time. We present Exemplar, a few-shot segmenter that fuses a frozen DINOv3 backbone with a fixed bank of classical native-resolution filter responses in one lightweight head, fitted from the support masks alone. In the few-mask, native-resolution regime, classical priors and frozen self-supervised features are complementary: fused in one head, a single fixed configuration spans eleven biomedical imaging datasets. Under the same head, the classical bank alone reaches 0.693 on the eleven-dataset panel, scored by foreground intersection-over-union or centreline Dice, and the frozen features alone 0.672; the bank leads on seven of the eleven and the features on the rest, and fused they reach 0.782. Against five forward-pass few-shot methods, Exemplar leads in 54 of 55 method-dataset comparisons, 52 of them significant after Holm correction. From a single annotated mask it reaches 0.703 on the same panel, against 0.682 for a from-scratch nnU-Net trained on that same mask. At eight masks nnU-Net overtakes it on the panel mean, chiefly on centreline agreement, but takes 16-77x longer to fit.

Estimating physical parameters from scientific images is a common inverse problem in materials characterization that often relies on expensive physics-based simulations. In electron microscopy, specimen thickness and crystal mistilt are critical parameters that govern how electrons scatter through the sample, and therefore the accuracy of any atomic-scale structure recovered from it. They are commonly inferred by matching experimental position-averaged convergent-beam electron diffraction (PACBED) patterns to simulated ones, but grid searches scale poorly and neural-network methods require extensive pretraining that may not transfer to new conditions. Here, we propose scalable Bayesian optimization of composite functions (SBOCF), a simulation-efficient method that exploits the known composite structure of the image-matching objective and the intermediate information contained in simulated images. By representing PACBED images with patch-level summaries and two correction terms, SBOCF preserves the original pixel-wise objective while reducing the number of modeled outputs from 24,649 to 11. Under a budget of 50 simulator evaluations, SBOCF outperformed standard Bayesian optimization with expected improvement on synthetic SrTiO3 benchmarks with thick and thin specimens, reducing the median final SSE by up to 290x in the thick-sample case. On experimental data, SBOCF produced parameter estimates consistent with previously reported values without task-specific pretraining. For a simulated mistilted specimen, using the SBOCF estimates in a downstream ptychographic reconstruction recovered sharp atoms that were otherwise blurred. These results establish SBOCF as a promising approach for inverse problems involving expensive simulators and high-dimensional structured outputs.

Automatic neuron reconstruction from light microscopy images is a central problem in computational neuroanatomy. While recent methods have achieved encouraging results on local image blocks, it remains unclear whether such progress translates to reconstruction that is both structurally accurate and scalable to the whole-brain scale. We present CORAL, the first benchmark for structure-aware evaluation of automatic neuron reconstruction from light microscopy images at both local and whole-brain scales. Built on a high-quality whole-brain fMOST dataset with carefully curated annotations, CORAL establishes two progressive tasks: block-level reconstruction, which evaluates reconstruction methods under limited spatial context, and brain-wide reconstruction, which assesses complete neuron reconstruction at the whole-brain scale. To account for topological correctness beyond geometric distance similarity, we introduce a structure-aware metric based on fiber prediction. To further achieve complete neuron reconstruction across the entire brain, we develop a brain-wide neuron tracing framework that extends arbitrary local reconstruction methods to the whole-brain scale through an iterative local-to-global process. Using this benchmark, we provide the first structure-aware comparison of mainstream methods for local neuron reconstruction and further evaluate their performance in brain-wide reconstruction. Our results underscore the importance of structure-aware evaluation and the need for more robust methods for complete neuron reconstruction.

High-resolution images of unprocessed surgical breast tissue can be obtained using microscopy with ultraviolet surface excitation (MUSE). This technique is considered a promising method for checking surgical margins during breast cancer surgery. In this study, MUSE images at 4x and 10x magnifications were compared using patch-level classification methods. Texture analysis (TA) based on local binary patterns (LBP) and deep learning (DL) with a base Vision Transformer (ViT) model were used. Both methods achieved similar performance at both magnifications. Using DL method, both 4x and 10x magnifications achieved 96.30% sensitivity, 100% specificity and 98.18% accuracy. Using TA method, 4x achieved better specificity (100% vs 93.33%) and 10x yielded higher sensitivity (100% vs 93.33%), but both had the same accuracy (96.67%). No clear improvement in performance was observed with 10x magnification. These results show that 4x imaging achieves the same diagnostic accuracy as 10x imaging. At the same time, 4x offers a larger field of view and faster image capture. Therefore, lower magnification can be effectively used in MUSE systems for accurate and efficient intraoperative margin assessment.

Hardware assurance relies on scanning electron microscopy (SEM) to verify nanoscale structures, but assembling the large, high-quality datasets required for automated analysis is impeded by time-intensive acquisition and strict intellectual property (IP) constraints on proprietary designs. We propose a privacy-preserving pipeline that secures IP by heavily distorting the functional design while generating a visually realistic synthetic dataset from a small set of initial examples. A StyleGAN first learns the distribution of hardware layout masks to generate novel, macroscopically varied structures. Subsequently, a conditional GAN (Pix2PixHD) translates these masks into realistic SEM images that preserve authentic textures and noise. The primary finding of this work is that a segmentation model trained exclusively on this synthetic data not only demonstrates a successful "sim-to-real" transfer to real images but also outperforms a baseline model trained on the limited real dataset. Because the underlying synthetic layouts are demonstrably novel and reproduce none of the specific proprietary routing of the original design, deploying the final segmentation model mitigates the risk of exposing sensitive IP to attacks like gradient inversion and membership inference, providing a highly secure, high-performance solution for hardware assurance.

Accurate 3D neuron segmentation in fluorescence microscopy is critical for neuroscience. However, the sparse and elongated morphology of neurons poses significant challenges to existing segmentation methods. These methods struggle to preserve both local details and global topology, leading to fragmented results. To address this, we propose NeuroRefiner, a multi-agent system that formalizes the human expert workflow involving iterative global observation and local editing. Specifically, NeuroRefiner comprises three collaborative agents dedicated to diagnosing topological errors, generating correction instructions, and validating refinement quality. To facilitate agent instruction-guided segmentation refinement, we propose TopoRefineNet, a dedicated 3D U-Net-based tool that leverages cross-modality feature fusion to generate refined masks. Through multi-round agent reasoning and voxel-level editing, NeuroRefiner produces topologically more accurate segmentations with enhanced interpretability. Experiments on the BigNeuron, CWMBS, and ZBFWB datasets demonstrate that NeuroRefiner outperforms state-of-the-art methods, notably achieving a 3.02% improvement in F1 score on the challenging ZBFWB dataset.

Malaria remains a leading cause of mortality in resource-limited settings, where expert microscopists are scarce. Automated diagnosis based on microscopy images thus has strong potential to improve care delivery. But for an algorithm to deploy, a necessary requirement is that it meet a suite of non-obvious (from a machine learning (ML) perspective) clinical constraints. Therefore, in close consultation with a national health center we developed a malaria diagnosis pipeline which addresses key requirements listed by the health care center but typically ignored in the ML malaria literature. In particular, it includes: (i) stopping criteria (to reduce image acquisition and time-to-result); (ii) human-in-the-loop functionality (for review and accountability); (iii) multi-species discrimination (since treatment varies by species); (iv) thick film detection (standard for microscopy); (v) computationally-efficient uncertainty calculations (to aid clinician review); and (vi) an edge device platform (since internet can be spotty in this catchment area). The mobile system performs all inference on-device using YOLOv13n deployed via TensorFlow Lite. It detects four species and white blood cells from Giemsa-stained thick blood smear images, aggregating per-image detections into slide-level parasitemia with World Health Organization (WHO)-standard quantification. This paper highlights these various clinical constraints and offers methods to address them. Evaluated on 2,739 annotated images across all four species, the system achieves [email protected] of 0.863, per-image parasite count correlation of r = 0.812, slide-level r = 0.951 (soft counting, 10 images/slide), and runs entirely offline with a pipeline time of 10.27 +- 1.65 s per image.

Image-based profiling captures rich phenotypic signatures for drug discovery and functional genomics. Large public datasets like JUMP Cell Painting now provide millions of images for systematic study. However, JUMP alone occupies 115 TB, and fragmented evaluation practices make systematic comparisons of representation methods impractical for many researchers. Here we present Nahual, an open-source framework for reproducible model deployment, and JUMP-lite, a 92.0 GB subset of JUMP that is approximately 1,250-fold smaller, selected to cover genetic modalities and compound annotations and reduced via lossy JPEG XL compression. Using these resources, we benchmark five representation methods, including classical features (CellProfiler) and deep learning models (MorphEM, OpenPhenom, SubCell, DINOv2). Moderate compression broadly retains signal relative to uncompressed images. Standardized phenotypic activity and consistency metrics reveal meaningful performance differences across methods. Together, JUMP-lite and Nahual provide a foundation for accessible, reproducible benchmarking of image-based cell representations.

Neuron counting and segmentation in microscopy images of neuronal cultures is a routine and time-consuming task in neuroscience research, traditionally performed through manual inspection or semi-automatic tools. We present NeuroAdaptTrainer, an open-source Fiji/ImageJ plugin that integrates a YOLO instance-segmentation model directly into the microscopist's workflow. The plugin allows a user to run automatic neuron detection on a single image or a batch of images, manually correct the resulting detections from within Fiji, and use those corrections to adapt the model to new imaging conditions via transfer learning. A built-in external validation module allows the base and adapted models to be compared quantitatively on a held-out annotated set. NeuroAdaptTrainer lowers the barrier for non-specialist users to benefit from deep-learning-based segmentation while keeping expert supervision at the center of the workflow.

Confocal microscopy of optically cleared and swelled tissue resolves complex biological structures in 3D, but such acquisitions are highly anisotropic: along the under-sampled axial direction the structure can appear discontinuous, hampering reconstruction and automated quantitative analysis. The usual remedy upsamples the axial dimension to an isotropic volume before training a segmentation model, which requires dense annotations in the upsampled space, a prohibitive labeling burden. We present an end-to-end, GPU-accelerated framework that overcomes this without additional annotations. The model is trained on the native acquisition volume; random rotation of training patches leverages the well-resolved lateral plane to supply the missing axial information, and a z-axis continuity loss keeps neighboring slices consistent. We adapt both a convolutional (3D U-Net) and a transformer (SwinUNETR) backbone, aggregate overlapping patches by Gaussian consensus, and compute point-spread-function-corrected membrane thickness by ray-surface intersection on the GPU. We apply the method to the glomerular basement membrane (GBM), a thin, highly convoluted part of the kidney's filtration barrier that grows more irregular in disease. Segmentation accuracy matches inter-expert agreement. Continuity-aware training improves reconstruction smoothness and suppresses a periodic terracing artifact at minimal accuracy cost. We quantify GBM thickness across the reconstructed 3D surface and capture disease-related thickening, enabling fully automated anisotropic 3D morphometry of biological structures without dense volumetric labels or image restoration.

Laser line-scanning microscopy enables fast volumetric imaging but produces anisotropic lateral resolution. Orthogonal line scans provide complementary directional information that can recover near-isotropic resolution, yet existing deep-learning methods require a separate model for each optical configuration. We present a unified, resolution-conditioned fusion framework based on Rank Enhanced Linear Attention (RELA). Feature-wise Linear Modulation (FiLM) conditions the network continuously on the resolving-power ratio, enabling one model to adapt across slit widths. We further introduce Adaptive RELA, which replaces fixed-kernel rank enhancement with ratio-conditioned multi-scale depthwise convolutions and uses a learnable attention temperature to adjust selectivity with degradation severity. Training data spanning multiple slit configurations are generated using a physics-grounded separable point-spread-function model verified against measured optical data at 48.3 dB accuracy. The resulting model achieves 34-40 dB PSNR across configurations, whereas unconditioned multi-slit training collapses to 24.3 dB and per-slit specialists lose 4-9 dB outside their training setting. It also generalizes smoothly to unseen intermediate configurations without interpolation artifacts. Ablations show that FiLM resolves configuration ambiguity, global linear attention captures long-range directional correspondences, and adaptive temperature yields an additional 2 dB in the challenging near-isotropic regime, where complementary signals are weak.

Optical microscopy of particle and fiber dispersions involves interpreting subtle visual cues influenced by specimen morphology, chemical composition, magnification, and illumination conditions. We introduce an artificial intelligence (AI) distillation framework that extracts semantically rich image embeddings from microscopy images using semantic anchors. A multimodal teacher combines each image's visual embedding with three text embeddings representing illumination modality, magnification, and specimen identity and morphology. Generated by LongCLIP's extended-context text encoder, this yields a 2304-dimensional block-structured teacher vector whose component blocks remain physically interpretable throughout training and inference. A student vision transformer (ViT) with a multi-layer perceptron (MLP) decoder is trained to reconstruct this teacher vector from the image alone, minimizing a mean absolute error (L1) loss that enforces coordinate-level fidelity to the teacher's block structure. A cross-entropy term over pseudo-classes derived from HDBSCAN clustering of the teacher embedding space acts as a collapse-prevention regularizer, enforcing inter-cluster separation without requiring contrastive negative mining. At inference, the student operates on image input alone, producing compact embeddings that recover the full semantic content of the teacher vector. The framework achieves approximately 80% pseudo-class validation accuracy and 75% Recall@1 on fine-grained specimen description labels under leave-one-out nearest-neighbor retrieval. These results demonstrate that semantic anchoring enables a vision-only student to acquire richer and more interpretable representations than image-only training, with direct applicability to retrieval, classification, and exploratory analysis of heterogeneous particle and fiber dispersions.

Data augmentations define the invariances learned by self-supervised learning (SSL). Standard augmentation pipelines were designed for natural images, yet scientific imaging modalities are governed by physical measurement processes with distinct symmetry and acquisition constraints. Enforcing invariances that contradict these constraints can distort learned representations and limit downstream performance, but practitioners moving from machine learning into a new scientific modality currently have little guidance beyond transferring natural-image pipelines unexamined. We address this gap with a principled, reproducible procedure for augmentation design in scientific SSL: we formalise the physics-aligned augmentation set as a union of measurement-consistent symmetries and acquisition-driven perturbations, and we give a concrete, largely label-free workflow---enumerate candidates, label each by the measurement operator, validate with representation-geometry diagnostics, and confirm by single-factor ablation---for selecting them. We instantiate the procedure for real-space electron microscopy and reciprocal-space 4D-STEM diffraction, and evaluate it across five SSL paradigms (DINOv2, SimCLR, MAE, VICRegL, I-JEPA) on classification and crystal-orientation regression. Physics-aligned augmentations substantially improve downstream performance for objectives relying on cross-view consistency, reduce geodesic error and improve robustness under realistic acquisition variability (detector gain, resolution loss), and systematically reshape representation geometry. While our experiments use electron microscopy, the procedure is modality-agnostic and applies to other measurement-driven domains such as medical and remote-sensing imaging. These results position augmentation design as a primary, and controllable, source of inductive bias in scientific self-supervised learning.

Understanding tissue organisation in multiplexed imaging requires modelling both cellular phenotypes and their spatial context. Existing approaches typically rely on handcrafted features, such as marker intensity statistics or cell-type proportions, which often fail to scale or generalise across cohorts with heterogeneous marker panels. We introduce MUL-T, a lightweight transformer framework that reframes tissue architecture as a masked contextual prediction task over discrete cell tokens. By learning contextualised [CLS] embeddings without task-specific supervision, the model captures higher-order cellular interactions while remaining computationally efficient. We evaluate MUL-T on several clinically relevant downstream tasks, including core-level tumour pattern classification, patient-level grading, PD-L1 positivity prediction, and cross-dataset treatment response prediction. Across tasks, MUL-T consistently outperforms classical feature-based baselines and achieves performance comparable to a foundation ViT model, despite substantially fewer parameters and lower training cost.