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Source image data for figures 1-6 and appendix figures S1-S9

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

This project contains four complementary datasets supporting Zhang et al. (2025). Dataset 1: Common Stocks (Welch Lab). Dataset 2: Shimkets Lab pretraining dataset. Dataset 3: Shimkets LS3934 nitrate experiments. Dataset 4: WT replicate dataset for aggregation fate analysis. Together these datasets cover the training, validation, and application phases of our deep learning framework for phenotypic quantification.

Organisms: Myxococcus xanthus

Human induced pluripotent stem cells (hiPSCs) are an attractive cell source for regenerative medicine. For its widespread use as a starting material, a robust storage and distribution system in the frozen state is necessary. For this system, managing transient warming during storage and transport is essential, but how transient warming affects cells and the mechanisms involved are not yet fully understood. This study examined the in uence of temperature cyclings (from−80°C to−150°C) on cryopreserved hiPSCs using a custom-made cryo Raman microscope, ow cytometry, and performance indices to assess viability. Raman spectroscopy indicated the disappearance of mitochondrial cytochrome signals after thawing. A reduction in the mitochondrial membrane potential was detected using ow cytometry. The performance indices indicated a decrease in attachment ef ciency with an increase in the number of temperature cycles. This decrease was observed in the temperature cycle range above the glass transition temperature of the cryoprotectant. Raman observations captured an increase in the signal intensity of intracellular dimethyl sulfoxide (DMSO) during temperature cycles. Based on these results, we proposed a schematic illustration for cellular responses to temperature uctuations, suggesting that temperature uctuations above the glass- transition temperature trigger the movement of DMSO, leading to cytochrome c oxidation, mitochondrial damage, and caspase-mediated cell death. This enhances our understanding of the key events during cryopreservation and informs the development of quality control strategies for hiPSC storage and transport.

Imaging Methods: recorded image

Organisms: Homo sapiens

External genital RGB images of conscious female ICR mice were recorded under normal fluorescent room lighting and annotated with estrous stage labels determined by vaginal smear cytology; these data were used to train and evaluate convolutional neural network models for non-invasive estrous staging.

Organisms: Mus musculus

Chromosome mis-segregation during meiosis in oocytes causes miscarriages and congenital diseases. Aging-associated premature chromosome separation is a major cause of mis-segregation. Effective prevention of premature chromosome separation has not yet been achieved. Here, we design protein-based artificial kinetochores that act as decoys to prevent premature chromosome separation. Designed artificial kinetochore-like decoys are submicroscale clusters of NDC80-NUF2-tethered protein particles that can establish a biorientation-like state by competing with chromosomal kinetochores for HURP-decorated microtubules. This competition reduces excessive bipolar microtubule pulling forces exerted on chromosomes, thereby effectively preventing premature chromosome separation during meiosis I and II in aged mouse oocytes. These effects suppress egg aneuploidy. This study provides a decoy strategy with biocompatible artificial kinetochores to prevent aging-associated meiotic errors in oocytes.

Imaging Methods: recorded image

Organisms: Mus musculus

The term polymethine dye (PMD) has been intimately linked to the dinuclear scaffold–two heterocycles linked together by a polymethine chain of varying length. Dinuclear PMDs have been a successful scaffold for non-invasive in vivo imaging in the biologically advantageous near infrared (NIR) and shortwave infrared (SWIR) regions of the electromagnetic spectrum. Trinuclear polymethine dyes, resulting from the addition of a third heterocycle into the polymethine chain, possess the same photophysical properties that make dinuclear dyes excellent fluorescent probes, but have yet to be investigated for in vivo imaging. Herein, we expand upon the dinuclear and trinuclear heptamethine scaffold by taking advantage of the increased reactivity of a cyclopentenyl linker and synthesize flavylium- and chromenylium-based SWIR-emitting fluorophores. The trinuclear scaffold instills the fluorophores with increased steric bulk, leading to beneficial photophysical properties in micelles and outperforming their classic dinuclear counterparts. In this work, we apply trinuclear PMDs for in vivo SWIR imaging in mice and find them to be particularly efficient at lymph node labeling upon intravenous administration.

Organisms: Mus musculus

Pheochromocytomas and paragangliomas (PPGLs) exhibit substantial molecular and immune heterogeneity, complicating risk assessment and treatment. Here, we define three distinct transcriptional subtypes (C1, C2, C3) through integrative transcriptomic and immunogenomic profiling. C1 is characterized by hypoxia-driven pathways and an immunosuppressive microenvironment, correlating with poor prognosis. C2 exhibits a highly inflamed immune landscape with robust CD8+ T cell infiltration, suggesting potential sensitivity to immunotherapy. C3 is enriched in metabolic reprogramming pathways and displays intermediate clinical outcomes. Genetic analysis reveals subtype-specific mutational patterns, with pseudohypoxic driver mutations (SDHB, VHL, SDHA, SDHD) predominant in C1 and C3, while kinase pathway alterations (NF1, RET) define C2. Single-nucleus RNA sequencing further delineates immune ecosystem diversity. Notably, we identify ANGPT2, PCSK1N, and GPX3 as key subtype-specific biomarkers, with ANGPT2 driving tumor progression in C1 and emerging as a potential therapeutic target. Our findings provide a refined molecular classification integrating immune and genomic features, offering a framework for improved prognostication and precision therapies in PPGLs.

Imaging Methods: bright-field microscopy

Organisms: Mus musculus

Programmed Cell Death Pathways Coordinate Neutrophil and Macrophage Clearance in Zebrafish and Are Differentially Exploited by Salmonella Typhimurium

Imaging Methods: fluorescence microscopy

Organisms: Danio rerio

Lung cancer is the leading cause of cancer-related deaths worldwide. Existing therapeutic options have limited efficacy, particularly for lung squamous cell carcinoma (LUSC), underscoring the critical need for the identification of new therapeutic targets. We previously demonstrated that the Transmembrane Serine Protease TMPRSS11B promotes transformation of human bronchial epithelial cells and enhances lactate export from LUSC cells. To determine the impact of TMPRSS11B activity on the host immune system and the tumor microenvironment (TME), we evaluated the effect of Tmprss11b depletion in a syngeneic mouse model. Tmprss11b depletion significantly reduced tumor burden in immunocompetent mice and triggered an infiltration of immune cells. RNA FISH analysis and spatial transcriptomics in the autochthonous Rosa26-Sox2-Ires-GfpLSL/LSL; Nkx2-1fl/fl; Lkb1fl/fl (SNL) model revealed an enrichment of Tmprss11b expression in LUSC tumors, specifically in Krt13+ hillock-like cells. Ultra-pH sensitive nanoparticle imaging and metabolite analysis identified regions of acidification, elevated lactate, and enrichment of immunosuppressive (M2-like) macrophages in LUSC tumors. These results demonstrate that TMPRSS11B promotes an acidified and immunosuppressive TME and nominate this enzyme as a therapeutic target in LUSC.

Imaging Methods: bright-field microscopy fluorescence microscopy

Organisms: Mus musculus

The CLK family plays a crucial role in regulating the phosphorylation of SR proteins. Therefore, to explore the dynamic effect of T-025 on the subnuclear localization of SRSF7 in triple-negative breast cancer cells, we performed time-lapse live cell imaging over 24 hours, with a 30-minute imaging interval. This was done using an established bacteria artificial chromosome (BAC) green fluorescent protein (GFP) Hs578T cell line that expressed SRSF7-GFP fusion product under the control of the endogenous promoter. BAC-Hs578T-SRSF7-GFP reporter cells were seeded with the seeding density of 15,000/well in glass-bottom Sensoplate 96-well imaging plate coated with 20 mg/ml rat tail collagen I in PBS. The following day, cells were stained with Hoechst-33342 and treated with 1 µM T-025. Live cell imaging was performed using a 20x objective (0.75 NA, 1.00 WD) on a Nikon Eclipse Ti microscope, equipped with a humidified 37 °C incubation chamber and 5% CO2 flow. Images were captured with a DS-Qi1MC CCD camera at 30-minute intervals for 25 hours, with 4 positions per well being recorded using NIS-Elements software.

Imaging Methods: confocal microscopy

Organisms: Homo sapiens

This dataset was acquired by confocal microscopy from environmental plankton tows during the TRaversing European Coastlines (TREC) expedition 2023-2024 (see Linked Information [1]). The dataset contains between 44 and 56 individual tiles for each of the 350 samples, acquired either in wide-field or confocal mode. The sampling sites and metadata are accessible via the respective BioSamples IDs. To check the scripts used for data processing, to open the data files and metadata of your choice, as well as to report any issue, check the supporting GitHub repository: https://github.com/AlexandraZakieva/live-confocal-super-plankton. NOTA BENE After the release of this accession, the community flagged key improvements that should be made. We will implement them in January 2026. Meanwhile, for the most updated version of the OME-Zarr files, have a look at the GitHub repository. The dataset is used for taxonomy analysis on the EcoTaxa platform at project ID 18361 [3]. Ontology according to Ontology Lookup Service [8]: Brightfield CHMO:0000104 Cell Wall GO:0005618 CalcofluorWhite CHEBI:50011 CFW CHEBI:50011 ChloA mesh:D000077194 Chlorophyll A mesh:D000077194 Chloroplast mesh:D002736 environmental material ENVO:00010483 Hoechst BAO:0000116 Hoechst33342 FBbi:00000052 Hoechst34580 mesh:C572112 PE mesh:D010799 Phycoerythrin mesh:D010799 polysaccharides CHEBI:18154 surface structures GO:0009986 T-PMT REPR:PMT

Imaging Methods: bright-field microscopy fluorescence microscopy confocal microscopy

Organisms: marine plankton environmental sample

Background Radiomics has significantly advanced radiation oncology by providing quantitative, objective metrics to predict therapeutic efficacy. The ability of radiomics to quantify intricate tumor characteristics enhances its potential role in precision oncology, particularly in predicting tumor response to treatments. However, these methods have not been applied to three-dimensional, multicellular tumor spheroids, which are the preferred in vitro model for pre-animal, pre-clinical selection of novel, future-oriented treatment modalities as they mimic the pathophysiological milieu of tumor microareas or micrometastases. Results: In this study, we implement an AI-driven predictive modeling workflow using radiomics data from early post-treatment imaging of spheroids of two human cancer cell lines subjected to radiation therapy and hyperthermia. The objective is to predict long-term tumor spheroid relapse from early post-treatment observations. For this purpose, we utilize a comprehensive dataset comprising image time series data from two distinct spheroid types across two independent experiments. We consider three post-treatment observation ranges - short, mid, and long - from which the treatment response is predicted. Our approach integrates multiple feature selection methods and machine learning algorithms for optimal classification performance. Our findings demonstrate that radiomics significantly improves the early prediction of tumor spheroid relapse. A detailed evaluation of the model performance reveals a time gain by early prediction of 2-14 days, while cases of late relapse remain challenging. Conclusions The presented radiomics-based approach potentially reduces the resource-intensive demands associated with prolonged experimental monitoring in a state-of-the-art spheroid assays and allows accurate prediction for up to three days beyond the observation horizon.

Imaging Methods: ANSOM

Organisms: Homo sapiens