Microscopy

Self-driving laboratories can explore synthesis conditions autonomously, but their decision-making layer is typically a black-box optimizer, and the output is a set of optimized samples, with the measurements reduced to predefined scalar objectives and the reasons behind success left unarticulated. Here we present SynAgent, a framework in which large language model agents operate an automated experimental system and maintain an explicit, revisable understanding of the synthesis process as the campaign's primary output. Starting with no predefined analysis pipeline, SynAgent adaptively generates analysis skills for newly acquired data and evolves this understanding through multimodal reasoning over experimental data such as X-ray diffraction patterns and electron micrographs. The evolution is guided by a verify-falsify scheme, in which the agent deliberately challenges its own hypotheses by testing conditions predicted to fail as well as those predicted to succeed. In a single campaign of 18 autonomous experiments using LiCoO2 (001) thin-film deposition as a testbed, SynAgent synthesized highly crystalline films and evolved an understanding of how the substrate temperature governs crystallization, discovering an abrupt threshold and a narrow optimal growth window at 650-690 °C. These results extend autonomous experimentation beyond optimized samples to testable, human-readable understanding.

Label-free clustering of frozen pretrained visual embeddings offers a scalable route to biodiversity monitoring, but image-only fine-grained taxonomy exhibits a consistent coarse-to-fine failure mode: clusters recover broad taxonomic structure yet plateau at species level. We study this behaviour on BIOSCAN-5M through an information-calibrated clustering analysis. BioCLIP~2 features with UMAP and HDBSCAN reach $0.79$ AMI at family and $0.67$ at genus, substantially improving over the prior image baseline and remaining competitive with oracle-$K$, graph-based, and learned clustering heads on the same frozen features. To diagnose whether the remaining plateau is method-limited or information-limited, we introduce InfoTaxa, which combines clustering efficiency---the fraction of probe-estimated image information recovered by an unsupervised partition---with paired DNA as an audit signal only, not an inference input. The density pipeline recovers approximately $0.90$ and $0.81$ of the image-available information at order and family, respectively. Held-out late-fusion probes show that adding DNA to the image embedding reduces species-level prediction error by approximately two bits. Robustness analyses cover multiple image encoders, described-species and rare-class subsets, probe diagnostics, and held-out-species coarse-rank generalisation and same-species retrieval. Thus, in the tested setting, species-level label-free clustering is both clustering-limited and representation-limited: improved clustering may recover additional image-exposed structure, but cannot close the DNA-audited information gap alone.

Characterizing two-dimensional (2D) quantum materials by optical microscopy requires localizing exfoliated flakes and determining their layer thickness from subtle optical contrast and interference color to select suitable flakes for device fabrication. However, models face synthetic-to-real domain shifts and variation across materials, substrates, laboratories, and imaging conditions. We present QuPAINT, a physics-aware multimodal framework for transferable quantum flake characterization. The Synthetic Materials Framework (Synthia) generates diverse synthetic microscopy images while preserving layer-dependent optical behavior. Using these images, we construct QMat-Instruct, a multimodal instruction dataset with image-specific reasoning traces generated from verified annotations and constrained to observable optical cues. QuPAINT integrates these signals through Physics-Informed Attention (PIA), which injects substrate-relative optical priors into the visual representation to support grounded multimodal reasoning. For evaluation, we introduce QF-Bench, to our knowledge, the largest real-world benchmark for this problem, spanning diverse microscopy and substrate conditions. Using its verified annotations, we study counting, visual grounding, reasoning quality, confidence calibration, and transfer to an unseen material. QuPAINT-8B substantially outperforms prior methods and establishes state-of-the-art performance for both general and monolayer flake detection. Additional experiments show that image-grounded supervision improves strict spatial grounding and confidence calibration while preserving robust general flake detection on the unseen material.

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.

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.

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.

Quantization is widely used to reduce the computational and memory demands of neural-network inference. In recurrent networks, however, the quantized state is stored and returned at the next time step, so the rule used to store that state can alter subsequent computations. Here, we introduce recurrent-state write-back to denote this rule and isolate its effect in a compact GRU encoder--decoder for fluorescence lifetime imaging, a molecular imaging modality used in quantitative biological imaging. A central task is estimating two lifetime parameters, the short-lived component {\tau}1 and the long-lived component {\tau}2, from high-noise time-resolved fluorescence signals. Holding the trained model fixed, replacing continuous state propagation with deterministic 4-bit state storage increases estimation errors for {\tau}1 and {\tau}2 by approximately 70x and 300x, respectively. Failure occurs when repeated small updates remain below the write threshold, leaving the stored state nearly fixed while the network continues to propose change. Error feedback, residual memory, and direction memory carry information from these suppressed updates across time and recover accuracy without retraining. Precision sweeps show that increasing state precision can worsen a fixed recurrent solution, while matched training shows that compatibility with the state interface can be learned. To test whether this behavior extends beyond the GRU, we repeat the post-training intervention in an independently trained LSTM, where coarse write-back reproduces the failure, error feedback restores accuracy, and state-specific interventions reveal greater sensitivity of the cell state than the hidden state. Our results establish recurrent-state write-back as a key determinant of low-precision recurrent dynamics and identify the state-storage interface as a central design consideration for quantized recurrent inference.

Surgical phase recognition is key to context-aware computer-assisted feedback in vitreoretinal procedures, yet the scarcity of synchronized multimodal intraoperative data, particularly microscope views and intraoperative OCT, limits approaches that aim to replicate the multimodal integration surgeons perform naturally. Surgical narration, by contrast, is abundantly available online and offers rich semantic supervision. Prior work has mainly explored pairwise contrastive learning (e.g., intraoperative OCT-microscope or microscope-narration), leaving the joint modeling of all three modalities largely unexplored. We introduce a framework that uses microscope views as a shared anchor to bridge surgical narrations and intraoperative OCT (iOCT) without requiring a fully synchronized tri-modal dataset, leveraging real microscope-narration videos and a synthetic dataset of synchronized microscope video and tool-aligned iOCT pairs. Contrastive alignment transfers structural priors from the synthetic domain to real videos lacking iOCT, and a dual-head MS-TCN++ integrates the resulting embeddings for joint macro- and micro-phase prediction. Evaluated on real vitreoretinal surgeries, our framework improves macro-phase recognition over a zero-shot baseline (mean F1 0.38 to 0.53) and provides an exploratory route to estimating fine-grained instrument-tissue measurements that are not directly observable in real microscope video alone; these micro-phase estimates are validated quantitatively on synthetic data and shown only qualitatively on real surgery. To our knowledge, this is the first work to unify microscope view, iOCT B-scans, and surgical narrations in a shared latent space for surgical phase recognition.

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.

Reliable three-dimensional (3D) navigation of magnetically actuated untethered microrobots remains a major barrier to clinical translation. X-ray fluoroscopy is the standard real-time imaging modality for endovascular procedures, but single-plane fluoroscopy provides only a two-dimensional (2D) projection, eliminating depth information and complicating autonomous navigation. Recovering this information through biplane imaging or repeated contrast-enhanced angiography increases procedural complexity, radiation exposure, or contrast burden. Here, we introduce VISTA (Virtual Integration for Spatial Tracking and Autonomy), a digital twin framework enabling contrast-free autonomous navigation under single-plane fluoroscopy. VISTA reconstructs vascular anatomy as a 3D digital twin, discretizes the vessel centerline into navigation milestones, and assigns the detected 2D robot position to the nearest projected milestone. Consecutive milestones define the local vessel orientation used to generate magnetic actuation commands, converting single-plane fluoroscopic observations into topology-constrained navigation states without requiring contrast injection during navigation. VISTA is demonstrated across anatomically distinct vascular phantoms under continuous flow and within the inferior vena cava of a live rat in vivo. Compared with conventional fluoroscopic human-in-the-loop control, VISTA reduced navigation time by up to 62%, corrective actuation commands by up to 98%, and radiation exposure by up to 57%. These results establish VISTA as a digital twin-guided framework for contrast-free autonomous navigation of untethered endovascular microrobots using widely available single-plane fluoroscopy.

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.

Leukemia cell image classification is challenged by real-world domain shifts from acquisition, staining, illumination, and site protocols, causing single-dataset models to generalize poorly in real clinical scenarios. This work presents a robust framework for leukemia classification across multiple heterogeneous datasets using a two-stage pipeline with a pretrained vision foundation model. Stage 1 performs binary classification (leukemia vs. non-leukemia) and is trained using 122,167 single-cell images. Stage 2 is conditionally applied to Stage 1 positives to perform subtype classification into Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML), trained using 69,400 single-cell images. Labels are harmonized across five heterogeneous datasets to enable cross-dataset training, and performance is evaluated on a held-out dataset protocol to assess domain-shift generalization. Within this pipeline, three encoders are benchmarked (DinoBloom, pretrained on single-cell images; BiomedCLIP, pretrained on biomedical data; and CLIP as a general-purpose model) under linear probing, Low-Rank Adaptation (LoRA), and a Retrieval-Augmented Classification (RAC) module that retrieves the top-k most similar cell images to provide cytomorphological grounding. The objective is to quantify how much domain-specific pretraining contributes to performance under domain shift, and whether cost-effective adaptation and retrieval can be a viable alternative to expensive domain-specialized pretraining. The held-out protocol additionally serves as a diagnostic tool, revealing when classification performance is attributable to dataset-specific artifacts rather than to cytomorphological features.

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.

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.

Scanning probe microscopy provides nanoscale access to structural, electrical, electromechanical, magnetic, and mechanical properties of materials. Its increasing use for wafer-scale characterization and combinatorial materials exploration creates a need to distribute measurements efficiently across large spatial domains. This is particularly important when available modalities differ in acquisition time and potential for tip and sample damage, making exhaustive multimodal mapping over spatial grids impractical. Here, we demonstrate multitask scanning probe microscopy, a live, closed-loop workflow in which a multitask Gaussian process learns spatial and cross-modal relationships and autonomously selects both the next measurement location and the next experimental protocol. The approach is implemented on an automated large-sample atomic force microscope and demonstrated on a composition-spread AlScN wafer using tapping-mode and Dual AC Resonance Tracking (DART) measurements. Paired initial measurements establish the relation between the tasks, after which noncoincident measurements are used to update both response landscapes. The resulting workflow extends active learning in scanning probe microscopy from spatial sampling to autonomous allocation of measurement modalities and provides a basis for combining rapid, weakly perturbative imaging with slower contact, electrical, electromechanical, magnetic, or spectroscopic measurements.

AI training's rising resource intensity is straining electricity supplies and carbon budgets, motivating systematic study of memory-efficient training on constrained hardware. We benchmark five gradient optimizers (SGD, Adam, Adagrad, Adadelta, and Conjugate Gradient Descent) under three memory strategies (standard training, gradient checkpointing, and gradient accumulation) across four transformer architectures (ViT, ModernBERT, Llama 3.1 1B, and NanoVLM), measuring training loss, GPU utilization, training time, and memory usage. Gradient accumulation emerges as the most reliable strategy, cutting training loss by roughly an order of magnitude on the vision-language model and about four-fold on the language model without additional GPU memory. Contrary to common practice, Adam is not universally superior: Adadelta and SGD outperform it on the encoder and autoregressive architectures. Gradient checkpointing's effect is strongly architecture-dependent, improving vision transformer loss while severely degrading the encoder model, and it increases training time by up to 60% on memory-bound models. GPU utilization is governed primarily by architecture, ranging from 8-15% for the memory-bound language model to 96-99% for compute-bound vision models. These findings provide practical guidelines for optimizer and gradient-strategy selection in resource-efficient model training and deployment.

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 mAP@0.5 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.

Fiber-bundle endoscopy offers a compact and flexible route for clinical fluorescence imaging through natural human orifices, but since its first report in the 1950s, it has remained limited by low spatial resolution, honeycomb artifacts, and inter-core crosstalk. The crosstalk becomes more pronounced at near-infrared-II wavelengths (NIR-II, 1000-3000 nm), a spectral window that offers superior contrast, resolution, and tissue penetration depth for biomedical imaging. Here, we present an AI-powered flexible endoscopy platform that overcomes these constraints through optical-computational co-design: optimizing ultrathin fiber bundles to mitigate crosstalk-induced image blur and enable high-fidelity image transmission across the visible-to-NIR-II spectral range, and developing an Agent-Guided Mixture-of-Experts (GAME) pipeline for honeycomb-artifact removal and image restoration. GAME provides a single restoration entry point for diverse biomedical images acquired with our endoscope, spanning cell, mouse and human samples. It dynamically routes each input to suitable restoration experts via a vision-language model, facilitating image reconstruction with a fourfold resolution improvement beyond the NyquistShannon sampling limit. The utility of our endoscope is demonstrated through in vivo NIR-II imaging of anatomical structures in mice, as well as imaging of the digital micromirror device (DMD)-projected human gastric tube and lymphatic system, paving the way for future clinical translation.

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.

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.

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.

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.

Objective: We evaluated whether metric 3D geometry of neurosurgical operative exposure can be recovered from standard monocular operating-microscope images combined with microscope pose data. Methods: In a phantom-based laboratory study, two aneurysm training phantoms were imaged with a ZEISS Pentero 800 microscope integrated with Brainlab Cranial Navigation. Microscope images from the standard composite video output were stored with synchronous microscope poses. After intrinsic and extrinsic calibration, depth was estimated with the pretrained Depth Anything 3 model without task-specific fine-tuning. Fused point clouds were converted to meshes using Poisson surface reconstruction. Reconstructions were compared with reference surfaces from structured-light scanning and fine-slice CT. Results: For phantom A, representing a deeper surgical corridor, reconstruction accuracy ranged from 1.95 $\pm$ 1.70 mm to 2.33 $\pm$ 2.15 mm. For phantom B, representing a directly exposed surface, accuracy ranged from 1.02 $\pm$ 0.93 mm to 1.52 $\pm$ 1.21 mm. Larger image sets mainly improved completeness, while accuracy remained within a narrower range. Corridor analysis showed preservation of overall geometry with local deviations in incompletely reconstructed regions. Conclusions: Standard monocular microscope images combined with navigation-derived pose data can reconstruct millimeter-range 3D surfaces using a foundation-model-based pipeline. These results show technical feasibility in a controlled phantom setting and support further development toward objective quantification of operative exposure, image fusion, and characterization of working spaces for future surgical instrumentation.

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.

Flame diameter is a key measurable parameter in microgravity droplet combustion, but its extraction from self-illuminated frames remains difficult because soot tails, blurred luminous boundaries, chamber reflections, and droplet drift introduce substantial measurement bias and operator dependence. This work presents an AI-enabled digital measurement workflow for automated flame diameter from combustion images. The workflow integrates automatic prompt-point generation into Segment Anything Model 2, employing Random Sample Consensus (RANSAC)-based circle fitting. The automatic prompt strategy removes subjective manual point selection, while the video memory mechanism maintains temporal consistency for drifting droplets, and the RANSAC fitting rejects soot-tail pixels as geometric outliers. The method is validated by 19,537 flame images of n-heptane, n-decane, and n-octane droplets with varying initial diameters. Compared with manual-reference measurements, the proposed workflow achieves a mean relative agreement of 96.9%, a mean absolute percentage error of 3.1%, and substantially outperforms conventional Hough circle detection, which performed worse under the same evaluation conditions. The results also show that the measurement accuracy improves with increasing droplet size. The proposed workflow has a combined standard uncertainty of 8.54% and achieves approximately a 229-fold improvement in efficiency over manual measurement. These results demonstrate that the proposed SAM2-based workflow provides a reproducible, fully automated, and metrologically characterized digital measurement system for extracting flame diameter from challenging combustion images. The approach supports high-throughput combustion diagnostics and illustrates that AI-based segmentation can be integrated into quantitative measurement workflows for digitalized image-based metrology.

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

Tree growth determines how much CO2 is sequestered from the atmosphere and temporarily stored in woody biomass. At the same time tree growth is affected by increasing temperatures, more frequent drought periods, late frosts and other extreme events associated with climate change. While continuous measurements of radial (secondary) tree growth using dendrometers are well established, monitoring of shoot elongation (primary growth) has largely been neglected because suitable measurement techniques are lacking. As a result, the effects of climate change on primary tree growth remain insufficiently understood. This work aims at reconstructing native deciduous trees in 3D as a basis for measuring and monitoring shoot elongation over entire tree canopies. Here we explored the use of low-cost UAV photogrammetry and of a multi-camera CraneCam system under real-world conditions. Data were collected in two study areas over an entire growing season. We present sensor evaluations, photogrammetric data acquisition and processing strategies. A special focus is placed on the analysis of the resulting photogrammetric 3D point clouds in terms of accuracy, resolution and completeness. Results demonstrate 3D point accuracies of 5-6 mm for entire trees using consumer-grade UAVs weighing less than 250 g and a 3D reconstruction completeness between 92% and 98% depending on the UAV type. The paper introduces a novel 3Dprinted ground-truth branch to evaluate the capability to reconstructing fine-detail structures such as thin tree shoots. Finally, we discuss operational challenges and initial experiments towards a skeletonization of entire trees based on photogrammetric point clouds.

Automated experimentation is moving from closed-loop optimization toward open decision-making, where human or AI planners must forecast the consequences of candidate actions before executing them. Such forecasts require a model of both sides of the experiment: how the sample is likely to respond and what the instrument is likely to detect. We therefore introduce a coupled digital-twin framework that separates these roles and then links them. In this framework, the sample twin encodes material state inferred from prior knowledge and measurements till the moment. The instrument twin captures signal formation, feedback dynamics, and operating constraints based on prior knowledge. When coupled, the two twins estimate expected outcomes, uncertainty, and risk for candidate microscope operations. For amplitude-modulation scanning probe microscopy, we realize this framework with a physics-informed encoder of force-distance curves, a deterministic scanner model of cantilever and feedback dynamics, and sparse learned residual corrections. The encoder first recovers scanner-driving descriptors with sub-nanometer accuracy. The calibrated scanner then reproduces typical traces within a few nanometers and identifies operating-point noise amplification as the main source of mismatch. Supplementary phase analysis localizes residual error to the phase channel, which clarifies where added physics is needed. Together, these results establish coupled sample and instrument twins as a practical foundation for predictive microscope operation and autonomous experimental planning.

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.

Mounted butterflies are among the most striking objects in natural history collections. However, their beauty is notoriously hard to digitize in 3D: they are small and fragile, with microscopic hairs and vein structures. Capturing them in sufficient detail, therefore, requires a macro lens, which has a very limited Depth of Field (DoF). Moreover, a camera body cannot be maneuvered beneath a pinned specimen to photograph its ventral surface (the underside of the wings). We introduce an end-to-end pipeline that resolves these challenges to turn such specimens into photo-realistic 3D models viewable from every direction. It combines three ingredients: handheld focus stacking for all-in-focus macro capture without a tripod, a non-contact first-surface mirror system that exposes the ventral surface without touching the specimen, and a segmentation-free, mirror-aware 3D Gaussian Splatting extension. We validate the reconstructions on four diverse specimens.

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.

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.

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.

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.

Deep learning in computational microscopy is severely constrained by the scarcity of densely annotated datasets. While synthetic data generation has bridged this gap in macroscopic computer vision, traditional graphics engines rely on geometric ray-tracing, failing to capture the micro-optical phenomena required for microscopy. Conversely, while wave-optics formulations exist, rendering them computationally tractable at the scale required for deep learning remains a massive systems challenge. To address this, we introduce the Optical Synthetic Object Generator (OSOG), a high-performance, fully differentiable forward-modeling engine. Drawing on established physical models of diffraction and phase retardation, OSOG maps continuous Optical Path Difference (OPD) calculations into a highly optimized, PyTorch-native Structure-of-Arrays (SoA) architecture. We validate this computational framework across three axes: First, object detection models (YOLOv11-OBB) trained purely on OSOG-generated data achieve robust zero-shot transfer to real-world highly occluded Lysozyme micrographs. Second, we introduce DiffOSOG, demonstrating that the engine's end-to-end differentiability allows for the exact recovery of continuous optical parameters via curriculum-guided inverse rendering. Finally, OSOG bypasses the $\mathcal{O}(N)$ bottlenecks of sequential ray-tracing, demonstrating sub-linear scaling by synthesizing 40,000 complex wave-optic particles in under 50 milliseconds (>20 FPS). By providing a fast, scalable, and physically grounded tensor pipeline, OSOG enables true real-time, on-the-fly dataset generation.

High-throughput microfluidic live-cell imaging generates rich single-cell data. Yet semi-automated procedures for locating regions of interest (RoIs), each containing one cell population, and removing surrounding microfluidic structures from recorded images, scale with the number of RoIs. This prevents real-time image analysis and delays time-to-insight by hours to days. We introduce the Design-Aware and Real-Time capable (DART) paradigm for microfluidic cultivation chips, which aligns the CAD blueprint with the physical chip and thereby enables throughput-independent localization of all RoIs and fully automated image processing across diverse RoI geometries and chip layouts. DART establishes this alignment through embedded fiducial markers and deep-learning-based marker detection. We validate DART using the Swiss Army Knife chip, which combines eight structurally distinct RoI designs across 1164 RoI locations. DART localizes all RoIs in five minutes, removes microfluidic structures from raw microscopy images in 40 ms, and performs fully automated image analysis, including cell segmentation, in under 1.1 s per image. Together, these capabilities establish DART as an end-to-end hardware-software paradigm with real-time-capable analysis that paves the way toward closed-loop and outcome-driven smart microscopy.

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.

Label-free, image-based cellular mechanophenotyping in microfluidic devices provides a high-throughput method for single-cell profiling. However, while complex microchannels (e.g., hyperbolic geometries) reveal transient deformation dynamics under continuous extensional stress, the resulting high-dimensional feature spaces are highly susceptible to hydrodynamic artifacts. Flow rate variations often distort discriminative boundaries, linking feature distributions to fluid conditions rather than intrinsic biology. To overcome this, we introduce a stability-guided analytical framework that decouples flow-induced noise from authentic mechanobiological signatures. We tracked the morphodynamic, kinematic, and intracellular optical-density trajectories of healthy and malignant ovarian cells to build a 93-dimensional feature space. Using a cross-flow screening strategy based on structural consistency and statistical persistence, we isolated robust descriptors, creating task-adapted subsets (20 features for binary classification; 25 for cancer subtyping). Variance-attribution analysis confirmed the neutralization of flow-conditioned artifacts; notably, flow-associated variance in the primary principal component fell from 69.9% to 9.3% in the subtyping task. We also found that macroscopic binary discrimination depends on bulk kinematic transitions, while clonal subtyping requires localized intracellular optical heterogeneity. These optimized subsets maintained diagnostic fidelity across multiple machine learning architectures and restricted sampling conditions. This framework establishes a robust, flow-independent foundation for continuous dynamic phenotyping.

A significant gap exists between theory and practice in deep learning. Generalization and approximation error bounds are often derived for simplified models or are too loose to be informative. Many rely on the manifold hypothesis and on geometric regularity such as intrinsic dimension, curvature, and reach. Progress requires insight into data-manifold geometry and suitable benchmarks, yet existing options are polarized: analytic manifolds with known geometry but limited applicability, or real-world datasets where geometry is only coarsely estimable. We introduce a benchmarking framework for studying data geometry. We repurpose and extend dSprites and COIL-20 with additional transformation dimensions and dense, axis-aligned sampling, and pair them with finite-difference estimators that recover curvature, reach, and volume at near-ground-truth accuracy in a regime where general-purpose estimators are unreliable or difficult to deploy. The framework is intended as a controlled testbed, useful as a calibration environment for geometric estimators and a sandbox for probing theoretical assumptions. To illustrate its use, we present two application studies, namely assessing the scaling behavior of the bounds of Genovese et al. and Fefferman et al., and tracking the layer-wise geometry of a $β$-VAE, highlighting the behavior of current bounds and the value of controlled benchmarks for guiding and validating future theory. A reference implementation is available at https://github.com/koulakis/manifold-microscope.

Many materials and chemical systems exhibit history-dependent responses, where functional outcomes are governed not only by final-state variables but by the time-dependent sequence of fields, temperatures, or chemical potentials applied during operation. Discovering new processing protocols is therefore a high-dimensional search problem in which the control variable is an entire waveform or sample history, and conventional strategies either remain confined to conservative interpolative families or become prohibitively measurement intensive. Here, a closed-loop workflow is introduced that couples evolutionary search over a compact waveform representation with uncertainty-aware deep kernel learning to generate, rank, and experimentally validate candidate protocols. Applied to ferroelectric thin films, with the scanning-probe tip-bias waveform as the protocol and the nonlinear electromechanical response as the reward, the workflow discovers waveform families that enhance nonlinearity by de-aging the film. Spatially resolved before/after measurements show that the best-performing waveforms selectively activate pre-existing, weakly pinned domain-wall segments, whereas the worst drive long-range irreversible switching. This framework reframes protocol tuning as out-of-distribution discovery, generalizable to synthesis and annealing trajectories, battery formation protocols, and other high-dimensional control problems.

Reflectance confocal microscopy (RCM) provides noninvasive, cellular-resolution "optical biopsies" of human skin \emph{in vivo} by acquiring en-face images at successive depths, forming a sparse z-stack. Due to optical limitations, these stacks are anisotropic 3D volumes with lateral resolution (0.5 $μ$m) $\sim$6 times higher compared to axial resolution, which is defined by the optical sectioning (3 $μ$m), limiting the interpretation of tissue. Our goal is to provide continuous-depth visualization by interpolating intermediate sections and making the 3D volume isotropic. Such a representation permits arbitrary-direction sectioning, including histopathology-like cross-sectional examination, without requiring per-patient optimization. To that end, we introduce the first RCM-specific novel-view synthesis (NVS) approach, CD-RCM, a feedforward model that predicts realistic, unseen depths from sparsely sampled RCM stacks. Classical neural rendering methods focus on reconstruction from surface-level multi-view observations. In contrast to surface-level camera views, RCM can acquire optically sectioned en-face images of tissue beyond the surface up to 200 $μ$m. However, during visualization of the RCM stacks, observations of the shallower sections (towards the surface) obscure the deeper ones. This unique axial imaging geometry and layer-dependent anatomical organization motivated our development of a tailored architectural and training framework that explicitly accounts for RCM's depth-resolved, occlusive imaging physics. Experiments demonstrate that CD-RCM achieves high-fidelity novel-view synthesis with sub-second inference time.

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.

In the production process of network cables, ensuring the correct color sequence of wire pairs inside the standard connector plays a critical role in the final performance of the cable, as any misplacement or color-ordering error can lead to defective products and impose significant costs. Traditional inspection methods based on visual examination through digital microscopes are typically time-consuming, tedious, and prone to human error. In this study, an intelligent system based on the twelfth version of the YOLO1 object detection model was developed to identify the position and verify the correct color sequence of wires in patch cords. The dataset used consisted of 2,500 images captured from microscopic views of network connectors, which were divided into 70% for training, 15% for validation, and 15% for testing. The proposed model, leveraging a single-stage architecture and attention mechanisms during learning, achieved highly accurate wire detection with approximately 98% precision. Additionally, the overall mean accuracy, classification precision, and recall were around 95%, 99%, and 98%, respectively. The results demonstrate that this system can reliably and in real time verify the correctness of wire color sequencing on the production line without the need for human intervention, thereby reducing human error and enhancing efficiency in the manufacturing process.

Single-Molecule Force Spectroscopy (SMFS) provides unprecedented insights into biomolecular mechanics, yet the high-throughput generation of force-extension trajectories creates a severe data curation bottleneck. Identifying rare molecular unbinding events within thousands of noise-dominated curves traditionally relies on tedious, non-scalable manual auditing. Here, we present a system-agnostic, interpretable deep learning framework tailored to overcome extreme class imbalance in automated SMFS triage. Utilizing 1D-to-2D rasterized geometric matrices, we deployed a modified ResNet18 architecture governed by an asymmetric Focal Loss objective function. We evaluated this framework on the complex mechanical unfolding pathways of the R. champanellensis cellulosome. Under hyper-imbalanced test conditions where the target interaction constituted only 1.34% of the dataset (13 true events out of 970 traces), the model achieved an overall accuracy of 0.9196 and a remarkable True Positive Rate (Recall) of 0.9231. By implementing an empirically calibrated dual-threshold triage system, the pipeline automatically discarded 880 unambiguous background noise traces , reducing the manual curation workload by over 90% while safely preserving high-value rare data. Finally, Gradient-weighted Class Activation Mapping (Grad-CAM) visually validated that the network's decisions are firmly anchored in the relevant geometric features of the force curves, specifically localizing on the structural unbinding regions, effectively mitigating 'black-box' skepticism. Built for free cloud-based execution, this open-source tool democratizes scalable, highly precise molecular discovery across the biophysics community.

Analyzing microstructural defects in transmission electron microscopy (TEM) images, particularly in irradiated metal alloys, is often limited by the availability of high-quality, labeled data. To address this, we introduce a generative data augmentation approach using a mask-conditioned latent diffusion model (LDM) for synthesizing realistic TEM images with controllable, automatically labeled multi-class defect masks. Without requiring manual annotations for generation, our method enables the creation of synthetic image-mask pairs by sampling distributions learned from experimental masks. These generated data were used to augment small experimental datasets of varying sizes (10, 50, and 100 labeled experimental images) to train a Mask Regional Convolutional Neural Network (R-CNN) model for defect detection and classification. Our results show that generative augmentation yields small overall model performance improvements, with up to a 0.02 gain in the harmonic mean of detection and classification F1 scores. However, we also find that the relative contributions to detection and classification improvement depend on the specific train/test data split. These findings highlight the potential of targeted generative models to enhance deep learning performance in data-scarce microscopy-based image quantification tasks.

Biological image analysis increasingly demands integration across heterogeneous tools, programming environments, and domain knowledge that few researchers can command simultaneously. We present Agentic-J, a containerised, multi-agent AI assistant, primarily for ImageJ/Fiji that enables biologists to specify analysis tasks in natural language, from nuclei segmentation and cell tracking to multi-condition quantification. The agent generates executable scripts organised into a documented project structure, so every analysis decision is traceable and the workflow can be reproduced or shared. The specialised sub-agents handle plugin management, code generation, debugging, quality assurance, and statistical reporting. In this paper we introduce the system's design, demonstrate real biological microscopy image analysis workflows, and detailed the technical implementation.