Exemplar: Classical Priors Complement Frozen Features for Few-Shot Microscopy Segmentation at Native Resolution

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.

QCell: Recombining and Aligning Cell Queries for Overlapping Instance Segmentation

Instance segmentation of overlapping cells in microscopy remains challenging due to semi-transparent structures that produce weak boundaries and mixed visual evidence in overlap regions. Existing methods address this through local regions of interest or shape priors but lack global reasoning across overlapping objects. We present QCell, a novel query-based model that de-overlaps cell instances in microscopy scenes. Our approach combines (i) an instance recombination module that decomposes and recombines query representations in latent space, enabling the model to reason about complete object structure under overlap, and (ii) a contrastive query alignment objective that combines distinctive instance feature learning and separation of overlapping cell queries. We additionally introduce a new Organoid dataset benchmark for overlapping cell segmentation. We show that QCell outperforms state-of-the-art methods across multiple benchmarks, achieving +2.2 AP and +2.7 AJI on ISBI2014. Code is available at https://github.com/SlavkoPrytula/QCell

XRF-to-Optical Field-of-View Localization with Vision Language Models

Registering images acquired with different microscopy modalities is essential for relating complementary measurements of the same specimen. In correlative X-ray fluorescence (XRF) and optical microscopy, the XRF map often covers only a small region of an optical image acquired from the same or an adjacent tissue section. Field-of-view (FOV) localization is necessary but can be difficult when appearance and structure differ across modalities. Here we evaluate training-free vision language model (VLM) localization on two datasets representing same-section high-correspondence and adjacent-section low-correspondence imaging. We test unconstrained and metadata-constrained search and compare VLMs with geometric controls, classical template matching, and two alternative training-free approaches (DINOv2 and multiGradICON). Direct VLM prompting produced content-dependent spatial signals but was not reliable alone. Classical matching was most accurate when cross-modal structure was preserved but failed in the low-correspondence collection. A proposal-and-verify workflow used repeated VLM predictions as candidates and image-based similarity to select the final location. This workflow recovered useful localization in the low-correspondence regime.

Conditional Neural Optimal Transport for Predicting Cellular Phenotypes from Molecular Structure

High-content microscopy enables systematic profiling of cellular responses to chemical perturbations, but the scale of the chemical space makes exhaustive phenotypic characterization experimentally infeasible. This motivates computational models that can predict image-derived phenotypes without acquiring the corresponding treated cells. We formulate molecule-induced phenotype prediction as an inductive conditional transport problem in image representation space. Given a negative-control phenotype and the structure of a molecule, we aim to predict the phenotype induced by the corresponding molecule. We first evaluate classical optimal transport baselines and show that static couplings do not yield useful predictions on large-scale phenotypic image datasets. We then introduce a molecule-conditioned Neural Optimal Transport (NOT) model with a Monge-Gap regularization training objective that learns to transport negative-control unperturbed phenotypes toward perturbed phenotypes using molecular structure as conditioning information. NOT recovers molecule-specific phenotypic effects while reducing microscopy-associated technical variation, thereby facilitating comparisons across experimental batches. On unseen active molecules, the model outperforms baseline approaches, demonstrating that chemically conditioned transport can generalize beyond the molecules observed during training. We identified the molecular encoder as the main limitation to this generalization, while transport in a compressed representation space improves performance and scalability. These results establish NOT as a promising framework for predicting cellular phenotypes from molecular structure and negative-control phenotypes, while highlighting the development of more informative molecular representations as a key direction for improving out-of-distribution performance.

Automatic Field-of-View Adjustment for a View-Expansive Microscope via LSTM-Based Gaze and Pipette Motion Interpretation

Intracytoplasmic sperm injection (ICSI) operators frequently adjust the field-of-view (FOV) during procedures, which interrupts workflow and increases procedure time. Conventional microscopes require manual objective lens switching and illumination adjustments to achieve different FOV sizes. We propose an AI-based automatic FOV adjustment method integrated with a view-expansive microscope. This microscope enables the simultaneous acquisition of a large FOV and high-resolution images using a single objective lens through multiview imaging with galvanometer mirrors and high-speed vision, thereby eliminating the need for physical lens exchanges. Our method utilizes a long short-term memory (LSTM) model to predict the appropriate FOV size based on real-time analysis of the pipette's position and velocity, combined with the operator's gaze position. The AI model is trained using ICSI procedure data from an expert with over five years of micromanipulation experience. Experimental evaluation with novice operators reveals that the proposed automatic FOV adjustment system significantly improves the ICSI procedure speed, reducing the average task completion time from 60.5 to 48.0 s (p < 0.001). The experiments also demonstrate that this improvement enables novice operators to achieve ICSI working speeds equivalent to those of expert operators.

NeuroRefiner: Morphology-Aware Multi-Agent Refinement for 3D Fluorescence Microscopy Neuron Segmentation

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.

Agentic self-driving microscopy benchmarks support qualification but do not necessarily generalize to unseen tasks

Large language model agents are increasingly being developed to control a wide range of scientific characterization tools including microscopes and synchrotron beamlines. Research into agentic control of physical infrastructure is nascent and there are few well-established paradigms for how to engineer an agentic system. There are many choices to make when designing a microscopy agent, including the choice of LLM, the number of agents to use, agent responsibilities and delegation rules, retrieval-augmented generation parameters, and more. When designing and optimizing an agentic microscope controller, researchers not only want to ensure that the agent can correctly perform known tasks but also that the agent can generalize to new tasks that it has not encountered before. In this study, we develop a benchmark and trace-logging framework that reveals a) how different choices of agent architecture impact performance at microscopy tasks and b) the limitations of benchmarks for predicting if a particular agent will perform well on unseen microscopy tasks. The framework was used to evaluate one-, two-, and three-agent graph topologies, five LLMs, RAG and context parameters, and operational constraints across 53 microscopy benchmark tests. In total, 105 agent configurations, 1,949 individual test runs, and 49,109 RAG retrievals were recorded. Direct comparisons showed clear differences in latency, token use, cost, and failure mode between configurations. However, surrogate models trained on agent architecture and test results did not reliably predict an agent's performance on new, unseen tasks. These results show that these benchmarks are useful for qualification, regression testing, diagnosis, and direct comparison, but the current heterogeneous test suite does not support a task-independent global configuration model.

A Unified Resolution-Conditioned Framework for Orthogonal Line-Scanning Image Fusion

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.

ZMIS-SAM: Segment Anything Model Enhanced with Wavelet Transform for Zooplankton Microscopy Image Instance Segmentation

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.

Investigating the Visual Cues of CNNs for Vascular Segmentation: A Case Study in Microscopy and Fundus Imaging

Vascular segmentation is a standard procedure for clinical diagnosis, yet the specific visual features determining model decisions remain poorly understood. This paper investigates the visual cues Convolutional Neural Networks (CNNs) use to segment blood vessels across two distinct imaging domains: fluorescence microscopy and retinal fundus photography. We employ a series of experiments to quantify the influence of shape, texture, and receptive field on segmentation performance. First, we isolate texture and intensity by evaluating performance on patches subjected to pixel shuffling and normalization. Second, we assess global shape relevance by training models on sparse contours and centerlines. Lastly, we quantify the required spatial context by systematically varying the network's theoretical and effective receptive fields. Within the scope of the evaluated datasets, we found that pixel intensity is more relevant than texture, though networks maintain surprisingly high accuracy even when both cues are removed. Furthermore, CNNs struggle to extrapolate full vessel geometry from shape cues alone, typically relying on a relatively small effective receptive field of around 20 pixels, though global context provides a modest benefit for fundus images. While specific to the modalities studied, this methodology offers a quantitative foundation to audit and refine deep learning systems in vascular imaging.

Physics-Informed Deep Learning Model for Cross-Modality Super-Resolution in Fluorescence Microscopy

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.

Video to All-in-focus Image Reconstruction Algorithm for Automated Microscopic Urinalysis

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.

HyperBank: A Differentiable Bank of Classical Priors for Few-Shot Spheroid Microscopy Segmentation

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.

Plug-and-Play Volumetric Reconstruction for Compressive Sensing Light-Sheet Microscopy

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.

SkelEM: Training-Signal Decoupling of Skeleton and Diffusion for Self-supervised Axial Super-Resolution in Volume Microscopy

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.

3D Masked Autoencoders are Robust Learners of Volumetric and Multimodal Cellular Representations for Microscopy

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.

PHOEBI: An Open-World Benchmark for Bacterial Identification in Phase-Contrast Microscopy

Optical microscopy enables rapid, label-free imaging of live bacteria and is the standard instrument for species identification across clinical, environmental, and industrial microbiology. Yet field samples are routinely polymicrobial and may contain organisms that were never seen during system training, and no computer-vision benchmark tests 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, paired with a leave-combinations-out (LCO) evaluation protocol that holds out entire species combinations to mirror the practical scenario of a model trained on catalogued mixtures that must generalise to unseen ones. On LCO, every gradient-trained per-image aggregator we test drops $0.39$ to $0.57$ F1 from the in-distribution to the held-out split, a systematic open-world recognition failure in the aggregator, not the visual representation. A linear probe of thirteen different encoders over the same features spreads only about six percentage points of F1 across general-purpose and biomedical pretraining objectives, confirming the representation is sound. We propose three lightweight $\textit{anchor-based}$ decoders that capture per-species presence geometrically over a shared frozen tile-feature pool, scoring $\textit{higher}$ on held-out combinations than on in-distribution validation.

CurvSegFlow: Time-Conditioned Flow Matching for Robust Segmentation of Curvilinear Structures in Noisy Biomedical Images

Accurate segmentation of curvilinear structures remains challenging in biomedical imaging due to their thin geometry, complex topology, and sensitivity to noise. This is particularly critical for microscopy images of cytoskeletal network, where low signal-to-noise ratios and dense filament crossings often lead to fragmented or inaccurate segmentation. In this work, we propose CurvSegFlow, a segmentation framework based on time-conditioned flow matching. Instead of predicting a segmentation mask in a single pass, the method models segmentation as a dynamic process that progressively refines a noisy initialization into the target structure through a learned velocity field. The proposed model combines a U-Net backbone with triple-term loss function and temporal embeddings to guide the refinement process across reconstruction stages. This formulation enables gradual error correction and improves the continuity of thin structures. CurvSegFlow is evaluated on multiple synthetic and real microtubule datasets, as well as on public benchmarks of retinal vessels, corneal nerves and coronary arteries. Across datasets, the method achieves competitive or superior performance compared to established segmentation models, with consistent improvements in precision and structural continuity, particularly under low signal-to-noise conditions. These results show that flow-based iterative refinement provides a robust and general framework for curvilinear structure segmentation. Overall, the proposed approach improves segmentation quality in challenging imaging conditions and generalizes effectively across modalities without architectural changes.

Million-scale multimodal pollen microscopy with expert-guided foundation models

Automated pollen identification from microscopy remains a bottleneck in aerobiology, palaeoecology and biodiversity monitoring, because scalable systems must generalise across specimen preparation, scanner settings and geographic origins while retaining palynological interpretability. To address this gap, we present a million-scale multimodal pollen microscopy resource, Pollen AI Atlas, assembled from pure-species whole-slide bright-field images spanning four geographic origins, four scanner settings and 46 taxon labels across 31 botanical families. Seeded by one manually selected exemplar per source slide, token-level mining and filtering produced 1,511,390 released grain detections with 99.6\% proposal precision in expert-curated test regions. Each detection was paired with machine-generated grain-level morphological captions from five open-weight vision-language models, guided by expert-verified palynological anchors, yielding structured descriptions of aperture systems, wall ornamentation, shape and size. Among the evaluated models, Gemma4 provided the most controlled primary caption set, combining tight length control, no leakage and the strongest text-retrieval performance. Baseline benchmarks with frozen visual features reached 88.16\% top-1 accuracy, while cross-regional retrieval showed that caption-derived text embeddings remained robust when image similarity degraded (mAP@20 0.811 versus 0.262). Released data, annotations, captions, splits, code, and weights provide a benchmark for pollen recognition, cross-regional domain adaptation and domain-specific multimodal microscopy learning.

AURA: Active-Response Attribution under Treatment Ambiguity in Bacterial Cytological Profiling

When a bacterial sample is exposed to several antibiotics, not every applied drug necessarily acts: if the organism is resistant to one of them, that drug leaves no morphological trace. The clinically meaningful quantity is therefore not which antibiotics were applied, but which ones were active. We show that these two are sharply decoupled in real E. coli microscopy - naively assuming the applied combination equals the active one is correct only about 37% of the time - yet existing computational tools are ill-suited to recovering the active set. Forward perturbation models such as scGen, CPA, and IMPA are designed to predict appearance from treatment, not the reverse, and inverting them degrades sharply; discriminative image classifiers tend to memorise strain- and batch-specific texture and fail to transfer across experimental replicates. We introduce AURA, which reframes the task as constrained, energy-based inverse attribution. Its central inductive bias is that the active set must be a subset of the applied set; this collapses the candidate space and lets AURA infer the active subset of applied antibiotics by decomposing residual morphology into antibiotic response atoms and selecting the subset with the lowest reconstruction energy, using no strain label at test time. AURA-E adds evidence-aware abstention, withholding a prediction when candidate explanations remain near-equally plausible. On cross-replicate transfer in an E. coli cytological profiling dataset, AURA recovers the active antibiotic combination with 95.47% exact-match accuracy.

CellNet -- Localizing Cells using Sparse and Noisy Point Annotations

Counting living cells is an important step in many biological research workflows. Our collaborators at the Wellcome Sanger Institute study vital genes in humans via large scale saturation genome editing screening, which requires repeatedly counting cells a great number of times. Computer Vision based automation is crucial for high throughput and resource efficiency. In this work, we develop a regression-based deep learning computer vision algorithm to detect and count cells in phase-contrast microscopy images. To reduce annotation effort, which in practice often becomes a bottleneck, we focus on counting cells only using sparse point annotations, which are fast and easy to acquire. By comparison to state-of-the-art 0-shot methods, we show that regression-based counting is a promising alternative in low data regimes. Through developing methods to automatically count living cells in microscopy images, we contribute to valuable research on the human genome. The code is available at https://github.com/beijn/cellnet.