Fluorescence Microscopy

Understanding fluorescence microscopy images is fundamental to automating biological research. However, existing methods typically learn predefined concepts from closed-set data, limiting transfer across tasks and biological contexts. In this work, we introduce MicroCLIP, a vision-language model that learns generalizable cellular and subcellular representations from natural language supervision for open-set image classification and retrieval. To pretrain MicroCLIP, we construct MicroPool-2M, a dataset of approximately 2.5 million fluorescence microscopy image-text pairs spanning diverse cellular structures, cell lines, and imaging conditions. Aligning microscopy images with biological concepts in natural language is challenging because most images lack descriptive captions and biological knowledge annotations. We therefore progressively establish image-text associations at three levels to provide MicroCLIP with richer semantic supervision: (1) category-level alignment links images to standardized labels defined by subcellular structure-cell-line combinations; (2) knowledge-level alignment incorporates biological priors derived from these labels to capture relationships across species and cell types; and (3) detail-level alignment uses a multimodal large language model to recaption and verify image-text pairs at scale, emphasizing fine-grained visual characteristics. Extensive experiments demonstrate that MicroCLIP achieves leading performance in zero-shot classification and text-image retrieval on microscopy images. It retrieves images with specific cellular characteristics from thousands of candidates and generalizes to unseen biological concepts without additional supervision. These results highlight MicroCLIP's potential as a foundation model for fluorescence microscopy.

The accuracy of detecting protein crystals for fluorescence microscopy images is very critical for high throughput and automated systems. Although the trace fluorescent labeling method could highlight protein crystals, reflection and emission from the fluorescence dye is not always due to crystal regions. Therefore, the analysis of the peak wavelength in the emission spectra of a fluorophore may not always yield effective results. In this paper, we show that using the subordinate color intensity corresponding to longer wavelengths than the peak wavelength of the emission spectra could improve the accuracy of protein crystal detection. Hence, we have built a segmentation method based on the percentile intensity of the subordinate color for trace fluorescently labeled (TFL'd) protein crystallization trial images. Compared to using the dominant color channel, our segmentation method on subordinate color channel was able to reduce the misclassification rate of likely-leads or crystals as non-crystals by the percentage of from 9.71% to 2.02% depending on the classifier. Similarly, the accuracy of classifiers were increased by the percentage of from 1.77% to 5.53%. Our method reached around 94% accuracy while keeping misclassification of likely-leads and crystals as non-crystals below 1%. Moreover, to evaluate the generalizability of our method, we have conducted new wet lab experiments on two proteins, Concanavalin A (Con A) and Ab inorganic pyrophosphate (AbIPPase), and the misclassification rate was below 1%. Our experiments show that using the subordinate channel may be more helpful for TFL'd protein trial image classification.

Dead cell detection in fluorescence microscopy is essential for phenotypic screening and drug efficacy assessment. However, automated identification remains challenging due to signal variability, optical artifacts, and colocalization ambiguities across multiple channels. Markers that define nuclear boundaries do not neatly coincide spatially or temporally with apoptotic markers. Furthermore, marker accumulation kinetics vary across individual cells and apoptosis stages, making universal thresholds unreliable. We present a systematic comparison of several computational approaches for detecting apoptotic cells in nucleus-stained specimens: (1) heuristic methods using intensity thresholds, distributions, correlations, and domain guidance; (2) classification using numerical features (Logistic Regression, Random Forest, XGBoost and LightGBM) and image features (ResNet-18 and EfficientNet-B0); and (3) anomaly detection, also using numerical features (Isolation Forest, one-class SVM and PCA reconstruction) and image features (convolutional autoencoders and Deep SVDD). Using a dataset of 258 images containing 65,527 cells/nuclei across multiple scenes with 243 manually annotated ground truth dead cells, we evaluate each method on metrics such as PR-AUC, Precision, Recall, F1-score, and macro-F1. Our results indicate that classification using image features (deep learning) and numerical features (traditional) achieved the best performance, with ResNet-18 and XGBoost significantly outperforming manually designed heuristics and anomaly detection methods. Our findings demonstrate that such biomedical problems remain highly challenging to solve even with the state-of-the-art computational and learning techniques. We aim to provide an effective decision-support tool to researchers while tackling such problems either fully-automatically or in a human-in-the-loop setting as an aide to domain scientists.

Fluorescence microscopy reveals where proteins localize, but only a limited number of proteins can be imaged in the same cell; generating these images from amino-acid sequence and the cell's morphological context enables in silico localization of unimaged proteins. The two conditions, however, play asymmetric roles: morphological context is spatially aligned with the target, whereas sequence is non-spatial and must specify protein-dependent localization within it, with recurring coarse patterns shared across proteins and finer protein-specific variation. Existing generators condition on both jointly, without separating what each explains. We introduce FACET (Factorized Asymmetric Conditioning for Efficient Transport), a probabilistic generative framework that encodes this structure as an explicit inductive bias: sequence semantics are learned from what context leaves unexplained, coarse localization regularities are shared across proteins through a semantic memory, and protein-specific variation is a bounded residual around them. A variance-preserving state projection further lets FACET perform continuous stochastic transport through a pretrained diffusion predictor with minimal parameter overhead. On held-out proteins, FACET improves spatial overlap by 34.3% on the Human Protein Atlas and 14.0% on OpenCell over a backbone-matched baseline, and reduces FID by 27.2% and 46.5%, respectively, with 75% fewer network evaluations. It also substantially improves protein-association structure recovery and yields better-calibrated predictions, while detailed ablations show complementary contributions from its design choices. These results identify factorized asymmetric conditioning, rather than generator capacity alone, as a key lever for high-fidelity, efficient, and biologically meaningful cellular image synthesis.

Poisson denoising of scientific data requires methods that account for signal-dependent noise while accommodating different data dimensionalities and preserving quantitative intensity information. We present BMND, a dimension-independent extension of block matching and collaborative filtering for Gaussian and Poisson observations. Building on the two-stage structure of BM3D and BM4D, BMND processes Poisson data directly, without a variance-stabilizing transform, by combining noise-aware patch matching with propagation of signal-dependent noise variances through collaborative filtering and aggregation. A dimension-independent reference-patch traversal scheme supports arrays with an arbitrary number of axes. An optional aggregation-aware mass conservation preserves the observed total intensity after weighted overlap-add. We evaluate the framework on one-dimensional physiological signals, two-dimensional images, and three-dimensional volumes, using controlled noise experiments and measured fluorescence microscopy acquisitions. The experiments demonstrate improved reconstruction quality from noise-aware matching and Wiener filtering, while low-count phantom experiments show reduced denoising-induced intensity loss through mass conservation. The framework provides a unified, non-learning-based approach to denoising across arbitrary data dimensions and is released as an open-source library.

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.

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.

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.

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.

Multiplexed fluorescence microscopy improves tissue segmentation by providing complementary channels including nuclear (DAPI) and membrane (E-cadherin), that together encode richer spatial context than single-channel imaging alone. However, multiplexed models require all channels at inference, limiting deployment where only a subset is available. This work proposes a cross-modal knowledge distillation framework that transfers semantic information from a frozen foundation model teacher processing multiplexed input to a lightweight student operating on the nuclear channel only. The distillation objective combines MSE-based probability matching, boundary-aware supervision, and learnable uncertainty weighting. SAM ViT-H and CellSAM are evaluated as teachers across four U-Net students: Swin-Tiny (27M), ResNet18 (11M), EfficientNet-B0 (5.3M), and MobileNetV3 (1.5M), on TissueNet and BBBC038. On TissueNet, the SAM-distilled Swin-Tiny student achieves Dice 78.36 (plus or minus 1.44), a 13.05-point improvement over the no-KD baseline (65.31 plus or minus 1.35) and 87.9% recovery of teacher oracle performance (89.12 plus or minus 1.21) at a 23x parameter reduction. KD consistently improves all four students by approximately 12 Dice points, confirming architecture-agnostic distillation. SAM ViT-H outperforms CellSAM as teacher across all settings. Cross-dataset evaluation on BBBC038 shows consistent gains without teacher retraining.

Light sheet fluorescence microscopy (LSM) enables high-resolution, three-dimensional (3D) imaging of biological specimens, providing rich volumetric data for studying cellular organization, pathology, and vascular networks. However, the size, dimensionality, and annotation burden of LSM data make supervised deep learning approaches costly and difficult to scale. Additionally, despite the abundance of unannotated LSM volumes, foundation models for this modality remain underexplored due to computational challenges and the complexity of volumetric representation learning. In this work, we introduce a 3D foundation model for LSM data, pretrained on a large curated collection of 3D images spanning multiple organisms, stains, and imaging protocols. We learn transferable volumetric representations by jointly optimizing for masked reconstruction and image-text alignment. The pretrained backbone drastically reduces the annotation burden, enabling efficient, few-shot adaptation for varied downstream tasks. We evaluate this approach on downstream segmentation, classification, and deblurring. Our results demonstrate consistent improvements over baselines, (1) when measured using standard evaluation metrics and (2) when rigorously assessed by domain experts. This highlights the potential of foundation model pretraining to reduce annotation requirements while improving performance across diverse LSM analysis tasks. Pretrained model weights and code for pretraining and finetuning are publicly available: https://github.com/AdinaScheinfeld/lsm_fm_public_repo.git.

Synapses are densely packed submicron structures that dynamically reorganize during learning and memory formation. Longitudinal in vivo imaging of fluorescently tagged synaptic receptors offers a promising opportunity to study large-scale synaptic dynamics and how these processes are disrupted in neurological disease. However, in vivo imaging with 2-photon microscopy uses low laser power and therefore suffers from low signal-to-noise ratio (SNR) and high shot noise, nonlinear tissue motion between days, nonstationary fluctuations in synaptic fluorescence, and significant blur induced by the microscope point spread function (PSF). Together, these factors make it challenging to detect and track synapses, especially in regions with high synaptic density. This paper presents a novel template-based framework for modeling synapses as varying luminance point sources that move under a nonlinear tissue deformation. Taking a unified Bayesian approach, we apply this model to microscopy data by deriving a posterior that incorporates a diffeomorphic mapping for domain warping, a Gaussian point spread function for the imaging process, and a Poisson observation model for raw photon counts. The Bayesian solution simultaneously: (1) Constructs a probabilistic template of synapse locations, (2) denoises and deconvolves the image data, (3) infers fluorescence intensities, (4) performs diffeomorphic image registration to correct for tissue motion, and (5) provides confidence regions for these parameter estimates. We demonstrate the framework on both a 2D+t simulated dataset and a 3D+t longitudinal in vivo microscopy dataset of fluorescent synapses imaged in a mouse over two weeks.

Accurate quantification of the geometry of curvilinear biological structures is essential for understanding cellular mechanics and disease-related morphological alterations. Microtubule curvature is a key descriptor of filament rigidity and mechanical perturbations. However, reliable curvature extraction from fluorescence microscopy images remains challenging due to noise, low contrast, and partial filament visibility. Existing approaches rely on segmentation pipelines with pre or post-processing, which are highly sensitive to segmentation errors and often fail under adverse imaging conditions. In this work, we propose MTCurv, a deep learning framework for direct, segmenta-tion-free regression of microtubule curvature maps from noisy microscopy images. Leveraging a synthetic dataset with pixel-wise curvature annotations, we reformulated curvature estimation as a regression problem and adapted an attention-based residual U-Net. To reduce hallucinations and enforce spatial coherence, we introduced a gradient-aware loss combining Mean Squared Error with a gradient consistency term. Beyond model and loss design, we evaluated commonly used regression and image quality metrics, revealing that many perceptual and blind metrics are poorly suited for curvature estimation. Correlation-based metrics, particularly Spearman correlation, emerged as more reliable indicators of curvature prediction quality. Experiments on two datasets of increasing difficulty demonstrated that MTCurv accurately recovers local microtubule curvatures, even in the presence of background fluorescence. Ablation studies highlighted the contribution of both residual encoding and attention-based decoding. Overall, this work provides a practical tool for filament curvature analysis and methodological insights for geometry-aware regression in biomedical imaging. Datasets and code are made available.

Structured Illumination Microscopy (SIM) enables rapid, high-contrast optical sectioning of fresh tissue without staining or physical sectioning, making it promising for intraoperative and point-of-care diagnostics. Recent foundation and large-scale self-supervised models in digital pathology have demonstrated strong performance on section-based modalities such as Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC). However, these approaches are predominantly trained on thin tissue sections and do not explicitly address thick-tissue fluorescence modalities such as SIM. When transferred directly to SIM, performance is constrained by substantial modality shift, and naive fine-tuning often overfits to modality-specific appearance rather than underlying histological structure. We introduce SIMPLER (Structured Illumination Microscopy-Powered Learning for Embedding Representations), a cross-modality self-supervised pretraining framework that leverages H&E as a semantic anchor to learn reusable SIM representations. H&E encodes rich cellular and glandular structure aligned with established clinical annotations, while SIM provides rapid, nondestructive imaging of fresh tissue. During pretraining, SIM and H&E are progressively aligned through adversarial, contrastive, and reconstruction-based objectives, encouraging SIM embeddings to internalize histological structure from H&E without collapsing modality-specific characteristics. A single pretrained SIMPLER encoder transfers across multiple downstream tasks, including multiple instance learning and morphological clustering, consistently outperforming SIM models trained from scratch or H&E-only pretraining. These results suggest that histology-guided cross-modal pretraining yields biologically grounded SIM embeddings suitable for broad downstream reuse.

In fluorescence microscopy, spectral unmixing aims to recover individual fluorophore concentrations from spectral images that capture mixed fluorophore emissions. Since classical methods operate pixel-wise and rely on least-squares fitting, their performance degrades with increasingly overlapping emission spectra and higher levels of noise, suggesting that a data-driven approach that can learn and utilize a structural prior might lead to improved results. Learning-based approaches for spectral imaging do exist, but they are either not optimized for microscopy data or are developed for very specific cases that are not applicable to fluorescence microscopy settings. To address this, we propose λSplit, a physics-informed deep generative model that learns a conditional distribution over concentration maps using a hierarchical Variational Autoencoder. A fully differentiable Spectral Mixer enforces consistency with the image formation process, while the learned structural priors enable state-of-the-art unmixing and implicit noise removal. We demonstrate λSplit on 3 real-world datasets that we synthetically cast into a total of 66 challenging spectral unmixing benchmarks. We compare our results against a total of 10 baseline methods, including classical methods and a range of learning-based methods. Our results consistently show competitive performance and improved robustness in high noise regimes, when spectra overlap considerably, or when the spectral dimensionality is lowered, making λSplit a new state-of-the-art for spectral unmixing of fluorescent microscopy data. Importantly, λSplit is compatible with spectral data produced by standard confocal microscopes, enabling immediate adoption without specialized hardware modifications.

Fluorescence-based Ca$^{2+}$-imaging is a powerful tool for studying localized neuronal activity, including miniature Synaptic Calcium Transients, providing real-time insights into synaptic activity. These transients induce only subtle changes in the fluorescence signal, often barely above baseline, which poses a significant challenge for automated synaptic transient detection and segmentation. Detecting astronomical transients similarly requires efficient algorithms that will remain robust over a large field of view with varying noise properties. We leverage techniques used in astronomical transient detection for miniature Synaptic Calcium Transient detection in fluorescence microscopy. We present Astro-BEATS, an automatic miniature Synaptic Calcium Transient segmentation algorithm that incorporates image estimation and source-finding techniques used in astronomy and designed for Ca$^{2+}$-imaging videos. Astro-BEATS outperforms current threshold-based approaches for synaptic Ca$^{2+}$ transient detection and segmentation. The produced segmentation masks can be used to train a supervised deep learning algorithm for improved synaptic Ca$^{2+}$ transient detection in Ca$^{2+}$-imaging data. The speed of Astro-BEATS and its applicability to previously unseen datasets without re-optimization makes it particularly useful for generating training datasets for deep learning-based approaches.

Multiplex immunofluorescence (mIF) enables simultaneous single-cell quantification of multiple biomarkers within intact tissue architecture, yet its high reagent cost, multi-round staining protocols, and need for specialized imaging platforms limit routine clinical adoption. Virtual staining can synthesize mIF channels from widely available brightfield immunohistochemistry (IHC), but current translators optimize pixel-level fidelity without explicitly constraining nuclear morphology. In pathology, this gap is clinically consequential: subtle distortions in nuclei count, shape, or spatial arrangement propagate directly to quantification endpoints such as the Ki67 proliferation index, where errors of a few percent can shift treatment-relevant risk categories. This work introduces a supervision-free, architecture-agnostic conditioning strategy that injects a continuous cell probability map from a pretrained nuclei segmentation foundation model as an explicit input prior, together with a variance-preserving regularization term that matches local intensity statistics to maintain cell-level heterogeneity in synthesized fluorescence channels. The soft prior retains gradient-level boundary information lost by binary thresholding, providing a richer conditioning signal without task-specific tuning. Controlled experiments across Pix2Pix with U-Net and ResNet generators, deterministic regression U-Net, and conditional diffusion on two independent datasets demonstrate consistent improvements in nuclei count fidelity and perceptual quality, as the sole modifications. Code will be made publicly available upon acceptance.

Accurate focus quality assessment (FQA) in fluorescence microscopy is challenging due to stain-dependent optical variations that induce heterogeneous focus behavior across images. Existing methods, however, treat focus quality as a stain-agnostic problem, assuming a shared global ordering. We formulate stain-aware FQA for fluorescence microscopy, showing that focus-rank relationships vary substantially across stains due to stain-dependent imaging characteristics and invalidate this assumption. To support this formulation, we introduce FluoMix, the first dataset for stain-aware FQA spanning multiple tissues, fluorescent stains, and focus levels. We further propose FluoCLIP, a two-stage vision-language framework that grounds stain semantics and enables stain-conditioned ordinal reasoning for focus prediction, effectively decoupling stain representation from ordinal structure. By explicitly modeling stain-dependent focus behavior, FluoCLIP consistently outperforms both conventional FQA methods and recent vision-language baselines, demonstrating strong generalization across diverse fluorescence microscopy conditions. Code and dataset are publicly available at https://fluoclip.github.io/.