Microscopy Image Analysis

Here is the plain text version optimized for arXiv's submission form. Custom macros (like \CV and \SI) have been converted to standard text/math so they render correctly on the webpage: Evaluating the fatigue life of structural steels conventionally requires mechanical testing lasting tens to hundreds of hours, making it impractical for rapid quality control. We present CV, a computer vision framework that estimates the fatigue life ($\log N_f$) of lightweight alloy steels directly from optical micrographs without physical testing.The pipeline features a seven-stage OpenCV preprocessing routine to remove artifacts, a 28-dimensional physics-informed feature extractor (quantifying crack morphology, grain structure, porosity, and texture), and a CNN regression model trained with a Gaussian negative log-likelihood (GNLL) loss to jointly predict $\log N_f$ and sample-specific uncertainty $\hatσ$.Evaluating three architectures (SE-CNN, ResNet-50, VGG-16) on a synthetic micrograph benchmark, ResNet-50 achieves $R^2 = 0.93$, RMSE = 0.18 log-cycles, and macro-F1 = 0.91. The GNLL objective reduces Expected Calibration Error by 76% compared to a mean-squared-error baseline (ECE: $0.089 \rightarrow 0.021$). Grad-CAM maps confirm the network attends to metallurgically meaningful microstructural features.Running in under 65 ms per image, the pipeline and synthetic dataset generator are open-sourced. Because validation relies entirely on synthetic micrographs, these results demonstrate methodological soundness under simulated conditions; a domain-transfer study on real field samples is the immediate next step.

As primary consumers in the marine food chain, zooplankton play a crucial role in maintaining marine ecological balance. However, the Segment Anything Model (SAM) exhibits limited performance in microscopic image instance segmentation due to its lack of zooplankton-specific domain knowledge. To address these challenges, we propose a novel instance segmentation model based on SAM and wavelet transform (ZMIS-SAM), effectively tackling issues such as inaccurate classification, discontinuous segmentation of slender appendages, and incomplete boundary segmentation. Our framework incorporates three core innovations: ZM-ViT enhances SAM's capability to model zooplankton morphology and image intensity distributions through two lightweight adapters, the Neighboring Feature Aggregation Module (NFAM) improves continuous segmentation of semi-transparent slender appendages by integrating general-purpose and domain-specific features, and the Wavelet-based Multi-scale Multi-directional Feature Enhancement (WM2FE) module effectively recovers high-frequency details to refine boundary segmentation completeness. Extensive experiments demonstrate that ZMIS-SAM achieves state-of-the-art instance segmentation performance on the zooplankton dataset and exhibits strong generalization capability across multiple public cross-domain datasets.

Electron microscopy enables nanoscale cellular visualization but faces a trade-off between imaging resolution and acquisition speed. Existing learning-based methods rely on single-stream architectures that struggle to balance perceptual realism and quantitative fidelity, either over-smoothing details or generating unrealistic hallucinations. This work introduces a frequency-adaptive dual-stream architecture to resolve this conflict. Using discrete wavelet transform, we decompose images into low-frequency structures and high-frequency details, then employ a conditional diffusion model for realistic global synthesis and a transformer network for precise detail recovery. Experiments on the EMDiffuse dataset show the method achieves superior LPIPS and resolution ratio, substantially outperforming existing approaches. The method also shows strong generalization across diverse biological samples, supporting fast and reliable electron microscopy imaging for structural biology and nanotechnology applications. The source code and associated dataset are publicly available to facilitate further research.

Cross-modality image translation offers a route to super-resolution fluorescence microscopy from low-resolution images while reducing phototoxicity and instrumentation demands. However, purely data-driven models can produce visually plausible outputs that are inconsistent with optical image formation. Here, we propose a physics-informed generative adversarial network for confocal-to-STED image translation that incorporates microscope-specific point spread function information into the training objective. Simulated and experimentally measured PSFs were evaluated using a limited paired confocal-STED dataset of TOM20-labeled mitochondria in human primary M2 macrophages acquired across different experimental days. Performance was assessed using reference-based and non-reference-based image-quality metrics, together with complementary frequency- and distribution-sensitive analyses. The no-reference metrics probed physics-relevant image properties, including spatial-frequency content, contrast, and signal-to-noise behavior. PSF-guided models improved structural fidelity, reduced local deviations, and achieved closer agreement with STED references than non-PSF baselines, particularly in frequency-domain analyses. These results demonstrate that optical priors can improve the structural fidelity and physical plausibility of generative microscopy models for cross-modality super-resolution imaging.

SWITi is a test-time method for reducing artifacts in tiled predictions, particularly for neural networks that learn posterior distributions from which solutions are sampled at inference time. Tiled predictions are unavoidable for large image data, and artifacts arise whenever tiles are smaller than a network's receptive field and when tiles are independent posterior samples. SWITi averages overlapping sliding-window predictions, so discrepancies between neighboring samples are spread across shifted tile positions rather than accumulating at fixed seam coordinates. For posterior models, SWITi uses no more tile samples than an MMSE estimate requires and therefore incurs no additional forward passes. Additionally, we introduce two reference-free metrics, the Fraction of Rejected Tests (FRT) and Artifact Severity (ASV), for detecting and quantifying tiling artifacts from a per-tile permutation test that compares the distribution of pixel gradients across tile seams against the surrounding image content. On pre-trained and published image splitting models across three fluorescence microscopy datasets in 2D and 3D, we show that SWITi substantially attenuates stitching seams while also improving reconstruction fidelity and resolution. Since tiling artifacts in posterior predictions can easily be mistaken for biological structures or for boundaries between biological structures, removing or reducing them using SWITi will improve the downstream processing of large image predictions, which is particularly relevant for biomedical data.

Single-cell light microscopy images have become an important data source for characterizing cell phenotypes, but their complexity and heterogeneity pose challenges to high-throughput automated analysis. Existing representation learning methods mostly rely on task-oriented modeling, which is limited by specific datasets and predefined tasks, making them difficult to generalize across different cell types and microscopy modalities, and experimental conditions. Although general-purpose methods have improved the generalization ability of image representation in recent years, their limited utilization of experimental background and biological context information still poses challenges in complex phenotypic analysis. Here, we propose scMIR, a vision-language foundation model for single-cell light microscopy image representation. By synergistically combining self-supervised image reconstruction with text-guided cross-modal alignment, scMIR can simultaneously encode morphological and biological semantic information in a unified representation space. scMIR is pre-trained on 207,957 image-text pairs, covering various cell types, microscopy modalities, and perturbation conditions. scMIR outperforms existing general models and task-oriented methods as systematically evaluated on various complex tasks using 16 benchmark datasets, including cell classification, clustering, phenotype inference, and batch effect correction tasks. Furthermore, scMIR shows a strong generalization ability across various tasks without requiring task-specific fine-tuning. With its unique advantages, we envision scMIR may promote the standardization and automation of high-throughput phenotyping workflows through supporting various downstream analysis tasks.

This study systematically benchmarks different data augmentation setups across the baseline YOLO26 model size variants to determine the most effective setup for adenovirus detection in TEM images. The benchmarked setups include NAS, GAS, GMAS, and DAS, all evaluated under identical training conditions. A modified YOLO26 model leveraging P2, expanded STAL, increased topk, and MuSGD was also tested across the same benchmarked setups. The adenovirus dataset, selected from the published TEM virus dataset, was re-annotated by leveraging adenovirus particle positions to generate YOLO-compatible bounding box annotations. The modifications produced their largest performance gains under GAS and GMAS, with modified YOLO26x trained using GAS achieving a mAP@50 of 0.80, Precision of 0.81 and a Recall of 0.80

Super-resolution can make inspection images appear sharper without preserving the evidence needed to detect a defect. We study this failure mode with a benchmark that separates reconstruction from detection and evaluates both at a predeclared low false-positive rate. Ten end-to-end repetitions combine independently generated line/space and contact-hole images with model training, calibration, clean controls, weak defects, and a held-out defect morphology. Every reconstruction is scored by the same local residual detector, while direct and jointly trained detectors form a separate comparison track. Reconstruction fidelity and inspection utility diverge: the two learned reconstruction models attain the highest structural similarity yet detect fewer defect pixels than bicubic interpolation in every paired repetition. A direct DeepLabV3 detector reaches $0.1984\pm0.0385$ recall at $0.000174\pm0.000084$ false-positive rate and satisfies the held-out feasibility criterion in all ten repetitions. An illustrative joint model, DPU-WaferSR, passes independent clean calibration but exceeds the held-out limit in all ten repetitions, demonstrating that calibration success does not guarantee transfer. Weak-defect recall remains near zero for every feasible method. Applying the unchanged policies to 4,591 public Carinthia-S masks further reveals large method-dependent shifts on real SEM texture. These results support a simple conclusion: super-resolution for inspection should be judged by preserved task evidence and operating-point transfer, not reconstruction quality alone.

Microscopic urinalysis is a routine diagnostic test at hospitals. Recent studies have demonstrated the effectiveness of deep learning methods to automate microscopic urinalysis. These methods rely on high-quality images of the urine samples in which each cell is clearly identifiable. However, in practice, the urine sample on a glass slide has a multi-layer structure; hence, all the cells are not clearly visible within the depth of field of a lens focused at a particular focal plane. It demands acquiring multiple images at different focal planes to correctly identify each cell in a given urine sample, which is a time-consuming task. In this paper, we propose to simplify the task by recording a video, in place of acquiring multiple images, while gradually changing the focus of the lens manually by hand. A typical length of the video is from 2 to 14 seconds. We reconstruct an all-in-focus image from the recorded video frames and apply a deep learning model to detect and classify urine sediments. As a proof of concept, we conduct experiments on 14 videos acquired by a trained lab technician in a usual diagnostic lab environment and show the effectiveness of the proposed automated urinalysis pipeline with our novel reconstruction algorithm.

We present NEEDL-Bench, a microscopy detection benchmark for Swiss Needle Cast (SNC), a fungal disease of Douglas-fir trees. Douglas-fir is a keystone species of major ecological and economic importance as a softwood timber resource, and SNC affects productivity by forming sexual reproductive structures (pseudothecia) that emerge through the gas exchange pores (stomata) of the needles, thereby blocking gas exchange and compromising needle function. To date, there is no dataset for automatic computer vision detection of these structures, despite computer vision being well poised to standardize and viably scale severity measurements. To address this, we present NEEDL-Bench, a dataset of 3250 annotated images from 1082 Douglas-fir needles, annotated for both keypoints and bounding-box detectors. This dataset exhibits a challenging collection of features, including blur, poor object contrast, small objects of interest, and occlusions. To better capture both the nominal distribution of the data and the full breadth of rare structures, we present two distinct evaluation splits: either random sampling from the collected images or sequential sampling to maximize structural diversity. We evaluate multiple popular keypoint and bounding box methods for detection on this dataset as a baseline and observe a maximum F1 score of 0.8479, suggesting significant potential for gains from future development on this problem. Further, we find that larger models generally do not show commensurate gains in performance on this dataset, indicating that improvements on this problem will not come from scaling laws but rather from domain-specific inductive biases.

Few-shot spheroid segmentation must adapt to new cell lines, microscopes, and illumination conditions from only a small set of annotated images. While foundation few-shot segmenters can be accurate, their large opaque backbones make it difficult to understand which visual cues drive success or failure. We study this question with HyperBank, a differentiable bank of classical image-processing operators combining Frangi vesselness, a Sauvola threshold pyramid, structure-tensor responses, gradient magnitude, and Laplacian-of-Gaussian filters. HyperBank is fitted on the annotated support images and evaluated on disjoint held-out images across three independently acquired spheroid datasets. We treat it not as a general replacement for foundation models, but as a compact, interpretable few-shot microscopy pipeline and an analytic-prior probe of which classical cues carry the few-shot signal. The results show that, adapted on the same few annotated support images, a compact bank of analytic priors is competitive with, and on small-cluster, contrast-driven data can outperform, much larger foundation models, while those models remain stronger on externally sourced, texture-dominated spheroids. Leave-one-family-out ablations indicate that the useful few-shot signal is distributed across operator families and strengthened by support-set-tuned morphology.

Artificial intelligence (AI) is transforming electron microscopy by enabling quantitative analysis of increasingly large and complex datasets for nanoparticle characterization. Recent advances in machine learning (ML) and deep learning (DL) have expanded microscopy from a descriptive imaging technique into a data-driven platform for structural interpretation, dynamic analysis, and scientific inference. This review examines AI methodologies for nanoparticle electron microscopy, focusing on transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), scanning transmission electron microscopy (STEM), and in situ TEM. The discussion is organized around the principal challenges in nanoparticle characterization, including particle detection, segmentation, morphology quantification, atomic-resolution restoration, defect identification, two-dimensional-to-three-dimensional structural inference, and analysis of dynamic processes in situ. We review computational approaches from conventional ML and convolutional neural networks to transformer architectures, self-supervised learning, foundation models, multimodal AI, and physics-informed learning. We further discuss integrating microscopy data with simulations, metadata, and autonomous experimentation to relate nanoparticle structure, dynamics, synthesis conditions, and functional properties. The advantages, limitations, benchmarking, and data requirements of current methodologies are critically assessed. Finally, emerging opportunities for foundation models, AI-guided microscopy, closed-loop experimentation, and autonomous materials discovery are discussed. By integrating advances across computer vision, materials informatics, and electron microscopy, this review highlights the role of AI in next-generation nanoparticle characterization and accelerated materials discovery.

Cleavage-stage embryo assessment in in vitro fertilization requires the integrated interpretation of cytoplasmic fragmentation, developmental stage, and blastomere symmetry. However, conventional visual assessment is affected by observer variability, particularly when fragmented regions are small, irregular, or low contrast. This study presents EMBRACE, a multi-task deep learning framework for jointly performing cytoplasmic-fragmentation segmentation, t2/t4 developmental-stage classification, and blastomere-symmetry grading from static cleavage-stage embryo microscopy images. EMBRACE combines a shared ResNet-50 backbone, a concatenation-based multi-scale feature-fusion (C-MSFF) module, a U-Net-style segmentation decoder, and two task-specific classification heads. After predefined inclusion and exclusion criteria, 9,137 annotated embryo images were divided into 7,309 training, 914 validation, and 914 held-out test images. On the held-out test set, EMBRACE achieved a Dice coefficient of 0.781 and an intersection over union of 0.677 for fragmentation segmentation. Developmental-stage classification achieved an accuracy of 0.995, macro-F1 of 0.994, and AUC of 1.000. Blastomere-symmetry grading achieved a balanced accuracy of 0.901, macro-F1 of 0.907, and quadratic weighted kappa of 0.859. These findings support the feasibility of combining spatially inspectable fragmentation localization with embryo-level morphology assessment in a single framework. External and prospective validation is required before clinical deployment.

In time-lapse fluorescence imaging, single-particle-tracking is a powerful tool to monitor the dynamics of objects of interest, and extract information about biological processes. However, tracked particles can be subject to occlusion and intermittent detectability. When these phenomena persist over a few frames, tracking algorithms tend to produce multiple tracklets for the same particle. In this work, we introduce self-supervised learning of visual features to compare tracked particles, and we exploit both visual and positional distances to robustly stitch tracklets representing the same particle. We demonstrate the performance of our stitching framework on time-lapse fluorescence sequences of Hydra vulgaris neurons. Results show high stitching precision, and reduction of errors made by previous algorithms on the same data by a factor of two.

Background and Objective: Quantitative analysis of cell dynamics is central to modern biological research, providing critical insights into immune cell interactions, disease progression, and drug mechanisms. Automated cell tracking in time-lapse microscopy remains challenging due to noise, morphological variations, overlapping cells, and dynamic events such as divisions and fusions. Methods: We present ARGUS, a framework for Accelerated, Robust, General, and Unsupervised Cell Tracking Solutions. ARGUS combines adaptive cell detection, dense Farneback optical-flow prediction, frame-to-frame linear assignment, and a sequence-level tracklet-refinement step that reconnects trajectory fragments across short temporal gaps. Results: On publicly available Cell Tracking Challenge datasets, ARGUS achieved detection accuracy of 0.905-0.971 and tracking accuracy of 0.897-0.964, with runtimes within 1 minute (5-6 seconds for 3 frames). Conclusions: ARGUS is a modular, interpretable framework that can be adapted to different imaging modalities and biological applications without training data or GPU infrastructure. The implementation is publicly available at https://github.com/Gitinc/argus

Male infertility contributes substantially to the global infertility burden, and sperm analysis remains central to diagnosis, treatment planning, and assisted reproductive technology. Conventional semen evaluation, however, is labor-intensive, operator-dependent, and limited by inter- and intra-observer variability, motivating the development of objective and reproducible computational approaches. This review provides a comprehensive and perspective-oriented synthesis of artificial intelligence-driven sperm analysis, with a focus on computer vision, deep learning, multimodal fusion, robustness, and clinical translation. We first review task-specific methods for sperm detection and counting, tracking-based motility assessment, semantic and instance segmentation, morphology and defect classification, functional assessment, and genetic integrity evaluation. We then summarize public datasets, benchmarks, evaluation metrics, and emerging multimodal strategies that integrate microscopic images, time-lapse videos, CASA-derived parameters, DNA integrity assays, and clinical metadata. Beyond algorithmic performance, we discuss key barriers to real-world deployment, including data scarcity, cross-center domain shift, annotation inconsistency, interpretability, uncertainty calibration, privacy-preserving learning, and workflow integration. Finally, we outline a staged clinical translation roadmap spanning technical standardization, multicenter retrospective validation, silent prospective evaluation, human-in-the-loop clinical testing, ART outcome validation, regulatory approval, and post-market monitoring. By organizing the field from task-specific visual recognition to trustworthy multimodal reproductive intelligence, this review highlights both the progress and the unresolved challenges required to translate AI-driven sperm analysis into clinically meaningful decision support.

Establishing large-scale, high-resolution neural connectivity maps is fundamental to elucidating the structural basis of brain function. However, when processing terabyte- or petabyte-scale electron microscopy data, over-segmentation inherent in automated reconstruction algorithms remains a critical bottleneck, requiring extensive manual proofreading spanning person-years. To alleviate the heavy reliance on annotated data and the limited flexibility of conventional tracing methods, we propose a training-free, targeted neuron tracing framework. Specifically, we introduce a skeleton-guided Heuristic Spatial Search paradigm that leverages geometric priors to iteratively reconstruct neuronal morphologies through a probing-verification cycle. To achieve robust zero-shot semantic verification, we further develop a Dimension-Aware Semantic Verification strategy built upon the foundation model NeuroSAM 2. This strategy resolves intra-slice splits via Planar Ensemble Consensus and inter-slice splits via Axial Spatio-Temporal Propagation. Notably, we integrate the proposed workflow into the Neuroglancer visualization platform, enabling an interactive human-in-the-loop proofreading system. Experimental results demonstrate that the proposed method outperforms supervised baselines and reduces manual proofreading time by 33.4%. The source code is publicly available at https://github.com/HeadLiuYun/Probe-EM.

Diagnosing and monitoring diseases frequently involves the analysis of human biological samples, with blood analysis being pivotal. Specifically, leukocytes, or white blood cells (WBCs), are essential markers for evaluating the body's defense mechanisms against infections. Traditional methods for WBC counting and classification are labor-intensive and prone to inaccuracies, primarily due to human error. The conventional processes for blood cell analysis, especially those concerning WBCs, are beset with difficulties. These include the laborious nature of manual counting and the susceptibility to errors, which can significantly impact the accuracy and reliability of disease diagnosis and monitoring. This study proposes an automated, machine learning-based solution aimed at mitigating the identified challenges. By employing a hybrid model that integrates Yolov5 for the detection of WBCs, coupled with a finely tuned, pre-trained MobileNetV2 model and a Logistic Regression classifier, the study innovates in the accurate identification, counting, and classification of WBCs into four distinct types. The methodology leverages the BCCD dataset for training and validation purposes. The application of the proposed hybrid machine learning model has yielded remarkable results, demonstrating a detection accuracy rate of 98% through the Yolov5 stage, and an unparalleled classification accuracy of 99.04% in subsequent stages utilizing MobileNetV2 and Logistic Regression. Additionally, Our proposed YOLOv5-based RBC detection module achieves an F1 score of 99.73%, which outperforms the baseline. These findings underscore the model's potential in transforming traditional laboratory practices for WBC analysis, offering a path towards more accurate, efficient, and reliable disease diagnostics and monitoring.

Hierarchical structure is common in image data, where fine-grained clusters often merge into larger, coarser semantic groups. In biological cell images, current self-supervised learning models often suppress this hierarchy, as coarse factors such as imaging modality can obscure finer morphological attributes in the latent space. We propose a hierarchy-aware self-supervised training framework to address this problem. Our method combines two components: a distillation framework with a segmentation teacher to improve morphological awareness in the latent space, and a hierarchy-aware contrastive loss based on HDBSCAN to improve decision boundaries between closely related subtypes at different hierarchical levels. Together, these components reduce the tendency of self-supervised learning to overemphasize coarse factors and instead align embeddings with semantic and morphological cues. This yields biologically meaningful sub-clusters driven by fine morphological detail. We train and evaluate our method on a curated corpus of 2.3 million single cells aggregated from 20 microscopy datasets, both labeled and unlabeled, covering 208 cell classes. Our method improves over baseline and counterpart methods, increasing average top-K accuracy by 2.8%, top-9 retrieval on the dataset with the deepest hierarchy by 6.3%, and downstream F1-score for biologically relevant drug classification from perturbed cell morphology by 7.8%.

We investigate volumetric reconstruction for compressive sensing light-sheet microscopy (CS-LSM), where fast volumetric imaging is achieved by encoding multiple axial planes into each camera exposure. To recover the underlying volume from highly multiplexed measurements, we propose a plug-and-play (PnP) framework that flexibly incorporates any user-specified denoiser into the reconstruction process. Building on a slice-based formulation, we further introduce an axial-coupled model that exploits correlations between adjacent slices to improve volumetric continuity. For efficient computation, we derive a Woodbury-based update for the data-consistency step in both the slice-based and axial-coupled formulations, and employ a Gauss-Seidel sweep for the denoising step in the axial-coupled model. Under a weakly convex regularization assumption, we establish subsequential convergence of the proposed algorithm. Experiments on synthetic and real zebrafish-heart data demonstrate that the proposed framework successfully recovers cellular structures from compressed measurements, and provide practical insights into the comparative performance of commonly used denoisers within the PnP framework under the CS-LSM setup.

Automated malaria diagnosis from blood smear microscopy is a critical challenge in global health AI; in resource-limited settings, the scarcity of expert microscopists remains the primary bottleneck to timely and accurate diagnosis. Three compounding failure modes prevent reliable clinical deployment of existing deep learning systems. First, end-to-end detectors treat unannotated cells as background during training, producing recall figures that are strongly influenced by annotation completeness rather than reflecting true cell recovery. Second, Non-Maximum Suppression tends to suppress valid detections in dense smear regions where infection counts matter most. Third, existing whole-slide detection pipelines lack per-cell spatial evidence for clinical audit, despite image-level explainability methods such as Grad-CAM having been applied to malaria image classification tasks. We present MalariAI, a two-stage decoupled framework that addresses all three failure modes in a unified pipeline. Stage 1 applies an annotation-agnostic distance-transform guided watershed algorithm to isolate every cell in a full 1600x1200 blood smear image, recovering 75.95% of ground-truth cells by centroid localisation across the 120-image NIH BBBC041 test set without any ground-truth input. Stage 2 fine-tunes EfficientNet-B0 with Focal Loss (gamma = 2.0, per-class inverse-frequency weights) on 64x64 crops, achieving 98.36% overall classification accuracy with 87.5% and 75.0% per-class accuracy on the rare schizont and gametocyte stages, compared to only 24.57% and 25.95% AP for a Faster R-CNN baseline on the same classes. Grad-CAM++ heatmaps generated per detected cell provide instance-level spatial evidence for clinical audit, enabling microscopists to verify model predictions at the individual parasite level without sacrificing classification performance.

Artificial intelligence is transforming our capability to solve biological challenges. In dimensionality bottleneck regimes exacerbated by high-dimensional biological data, neural networks force distinct concepts into the lower dimensions known as superposition. Although this superposition is widely known to hinder interpretability, its impact on corrupting the geometry of latent spaces remains critically overlooked. Here, we utilized sparse autoencoders (SAEs) trained on over 100,000 multiplexed images of patient-derived Parkinson's disease and healthy neurons to resolve superposition. This approach bypasses the mathematical non-uniqueness of feature attribution by shifting to interpretable latent representation analysis. We theoretically and empirically demonstrate that superposition contaminates representational metric spaces, and thereby SAEs successfully recover geometric fidelity. By treating these geometrically purified representations as single-cell state vectors, we adapted single-cell RNA sequencing (scRNA-seq) data analysis methodologies directly to the image domain. Finally, we introduce GW-map, utilizing Gromov-Wasserstein optimal transport to align these image representations with authentic scRNA-seq data de novo. This coupling reconstructs hierarchical neuronal pathology pathways such as Calcium-AIS scaffold, without reference spatial transcriptomics, establishing a scalable foundation for spatial biology. Code is available at https://github.com/jijihihi/Bio\_superposition

Volume microscopy, including electron and light microscopy, suffers from severe anisotropic resolution due to physical axial sectioning. Existing self-supervised axial super-resolution (ASR) methods face a trilemma bounded by overly smoothed regression textures, structural hallucinations of pure diffusion models, and prohibitive inference latency. In this paper, we propose Skeleton-refinE Microscopy (SkelEM), a self-supervised framework that decouples ASR at the training-signal level: a frozen topological network and a diffusion refiner are optimized by disjoint objectives, separating low-frequency topology formulation from high-frequency detail enhancement. Building on this deterministic skeleton, we exploit a unified cycle-consistent mechanism on input sparse slices to simultaneously extract a real-domain residual prior and bidirectionally align the diffusion refiner, washing away cross-plane artifacts without synthetic bias. By truncating the reverse diffusion process with this physical prior, SkelEM achieves high-fidelity detail restoration in merely $\le 5$ steps. To rigorously assess cross-instrument generalization, we further introduce BRAVE-ASR, a new benchmark of co-aligned anisotropic and isotropic volumes acquired on a Plasma-FIB instrument. Across public benchmarks, SkelEM achieves the most favorable balance across the fidelity-perception trade-off among self-supervised methods, with state-of-the-art downstream membrane segmentation performance and robust zero-shot generalization across distinct modalities.

A central premise of autonomous scientific imaging is that smarter navigation, whether Bayesian, RL-based, or otherwise adaptive, is the principal lever for sample-efficient acquisition. We present evidence to the contrary in scanning transmission electron microscopy (STEM), an atomic-resolution imaging modality whose every measurement deposits damaging electron dose. We introduce STEMGym, an open-source Gymnasium benchmark of 15 physics-simulated STEM worlds spanning five materials, three difficulty levels, and four characterisation tasks, scored by the Dose-Efficiency Curve area (DEC-AUC), a single scalar capturing the information-vs-dose Pareto frontier. Across 33 agent configurations under realistic dose budgets, the dominant determinant of dose efficiency is the analyst (perception) pipeline, not the navigator: pairing a trained CNN analyst with naïve raster scanning raises DEC-AUC by 5.5x over a CNN-free raster baseline (0.287 vs.\ 0.052), while substituting Bayesian or adaptive finite-state-machine navigation for raster yields no statistically significant further gain. Production-tier vision-language models further underperform task-specific CNNs by {\sim}13x on crystallographic defect analysis. By decoupling perception, navigation, and planning under a unified dose budget, STEMGym reframes where ML effort should be invested in autonomous electron microscopy and provides the measurement infrastructure to test it.

This work examines perturbation generalization in spatial foundation-model embeddings derived from fluorescence microscopy images. Although these models can discriminate drug conditions accurately, it remains unclear whether the learned representations reflect patterns consistent with expected perturbation axes that transfer across drugs. We introduce SVC-Probe, a perturbation-aware framework that combines Subcellular Embedding Atlas Stability, Mondrian Neighborhood Graphs, and a Foundation Model Perturbation Probe to assess embedding stability, neighborhood rewiring, and centroid prediction under drug treatment. Applied to the CM4AI MDA-MB-468 chemical-perturbation atlas comprising 462 antibody labels and SubCell 1536-dimensional embeddings, SVC-Probe demonstrates that 98.6% three-way condition accuracy does not correlate with reliable cross-drug prediction, with cosine similarity diminishing from 0.944 in-domain to 0.30 under leave-one-drug-out evaluation, constituting a two-drug stress test rather than a general benchmark. Null calibration indicates that raw residual-turnover coupling is largely influenced by generic embedding structure, whereas a drug-specific signal emerges under vorinostat and is consistent with chromatin-related reorganization. In contrast, the paclitaxel axis is not robustly reconstructed, likely due to sparse coverage of microtubule-associated proteins. Together, these results introduce and demonstrate a reusable diagnostic framework for stress-testing spatial virtual-cell representations and indicate that perturbation generalization may serve as a stricter and more informative benchmark than baseline condition discrimination.

Fluorescence microscopy images are degraded by noise and diffraction-induced blur, which compromise structural fidelity and limit quantitative analysis. Supervised deep learning methods achieve impressive restoration performance but require large-scale paired datasets that are difficult to obtain in practice. To address this issue, we propose SDIP, a zero-shot deep image prior (DIP) framework that sequentially performs denoising and deconvolution without external training data. An aSeqDIP-based module first suppresses noise while preserving fine structures through sequential autoencoding regularization. In the deconvolution stage, a wavelet-based background correction step is incorporated before the proposed RLG-DIP module performs artifact-reduced deconvolution. RLG-DIP uses the Richardson-Lucy deconvolution result as a physically consistent guidance prior, integrating the imaging model with the implicit prior of DIP to stabilize the ill-posed deconvolution process. Experiments on the BioSR dataset across multiple cellular structures demonstrate that SDIP improves both signal-to-noise ratio and resolution, achieving superior visual quality and improved quantitative performance on most evaluated structures. The proposed framework may also provide useful insights for designing physically guided DIP methods for other inverse problems.

Diffusion MRI (dMRI) tractography enables non-invasive reconstruction of white-matter pathways, but its accuracy is fundamentally limited by indirect, low-resolution measurements of axonal organization. Tracer injection studies in non-human primates provide a gold standard for validating dMRI tractography. This, however, requires time-consuming manual annotation of fiber bundles in histology sections. We propose a synthetic-data augmented framework for automated fiber bundle segmentation in macaque tracer histology. Our approach uses ex vivo dMRI tractography as a generative prior to synthesize 2D image patches for training. This provides us with sufficiently realistic foreground texture, which we compose with backgrounds from blockface photos and diversify via domain randomization. A 2D U-Net is trained on mixed real and synthetic patches. Experiments on held-out brains demonstrate improved generalization across brains and fiber bundle densities compared to training with real data only. Training with synthetic data only leads to poor performance, underscoring the need for real supervision. Overall, our approach achieves performance comparable to the state-of-the-art while requiring 3x less manually annotated data.

Self-supervised learning in fluorescence microscopy often relies on 2D projections, despite the inherently three-dimensional nature of cells. We present a systematic comparison of 2D and 3D masked autoencoders (MAE-2D vs. MAE-3D) on volumetric microscopy data. Under matched architectures and training protocols, MAE-3D consistently outperforms 2D max-projection and slice-based variants on downstream single-cell tasks. We further align visual representations with a pretrained protein language model (ESM2) and show that cross-modal supervision yields larger gains for volumetric models. Channel cross-attention and frequency-domain regularization are critical for leveraging 3D spatial context. On protein--protein interaction prediction, our best model achieves a ROC--AUC of 0.86, while on protein localization it reaches an AUC$_{\text{micro}}$ of 0.95 and an F1$_{\text{micro}}$ of 0.74, demonstrating competitive performance on both tasks. Overall, our findings highlight the potential of volumetric modeling and multimodal alignment for representation learning in single-cell microscopy.

Optical microscopy (OM) enables rapid, label-free imaging of live bacteria and is the standard instrument for species identification across clinical, environmental, and industrial microbiology. Real samples, however, are routinely polymicrobial and may contain organisms never seen during training, and no computer-vision benchmark evaluates multi-label species identification from phase-contrast microscopy (PCM) of such mixtures. We introduce Phase-contrast Optical bEnchmark for Bacterial Identification ($\textbf{PHOEBI}$), a wet-lab-prepared dataset of $120{,}000$ PCM images covering $40$ combinations of six rod-shaped species, together with a leave-combinations-out (LCO) protocol that holds out entire species combinations, mirroring a model trained on catalogued mixtures that must recognise new ones. Under LCO, gradient-trained per-image classifiers, from fine-tuned backbones to attention-based multiple-instance learning, collapse on unseen combinations despite high in-distribution accuracy, and the failure lies in how per-image predictions are aggregated rather than in the visual representation. We propose three lightweight $\textbf{anchor-based}$ decoders that read each species' presence against fixed geometric prototypes over a shared frozen tile-feature pool, and they remain stable under the same shift. Without additional training, the same features also support open-set rejection of unseen species and the discovery of a new class from unlabeled test images, with negligible disruption to the known classes.

Microscopic imaging provides essential visual evidence for studying plant biology and pathology at the cellular and subcellular levels. However, existing benchmarks on vision-language models primarily focus on macroscopic plant imagery, while the microscopic domain remains underexplored. To address this gap, we present PlantMicro, a comprehensive benchmark for evaluating vision-language models (VLMs) in microscopic plant imagery. PlantMicro integrates more than 5,000 images collected across diverse hosts, biological domains, and imaging modalities. Building on this diversity, we design a set of complementary tasks that capture different facets of microscopic image understanding. To support these tasks, we construct over 9,000 VQA pairs that systematically evaluate the capabilities of VLMs. Experiments on PlantMicro show that current VLMs struggle with fine-grained recognition and biologically grounded reasoning. For example, GPT-5 achieves 34.93% accuracy on the pathogen classification task, which is only modestly above the random-guessing baseline. The results highlight a significant gap in current VLMs' ability to comprehend plant microscopic images. PlantMicro provides a standardized foundation for advancing VLMs toward reliable and comprehensive microscopy-level plant understanding.