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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

Volumetric calcium imaging datasets obtained through scanned temporal focusing 2-photon microscopy at the snout region of the main motor cortex of awake passive mice. See methods section from https://doi.org/10.1101/2025.06.17.660119 for more details.

Imaging Methods: two-photon laser scanning microscopy

Organisms: Mus musculus

Cancer-associated fibroblasts (CAFs) are critical components of the tumor microenvironment (TME), yet their spatial and functional heterogeneity remains incompletely defined. Here, we constructed a comprehensive pan-cancer single-cell and spatial proteomic atlas integrating 461 patient-derived single-cell transcriptomes and 72 CODEX/PhenoCycler-Fusion–imaged tumor samples across eight solid cancer types. Using spatial multiplex imaging, we mapped the localization and immune interactions of distinct CAF subtypes, including myeloid-associated and T cell–associated inflammatory CAFs (iCAFs). Our data reveal that Mac2_iCAFs co-localize with M2 macrophages in hypoxic niches, while Tem_iCAFs associate with cytotoxic T cells in immune-enriched regions. These spatially resolved datasets provide mechanistic insight into fibroblast–immune crosstalk and serve as a resource for investigating stromal remodeling across human cancers.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

Screening of a comprehensive collection of 1,920 S. aureus mutants (Nebraska transposon mutant library) in epithelial cells across five timepoints (0.5, 1.5, 3, 6 and 48 hours post-infection) using a fluorescence microscopy-based infection assay.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

In order to study eIF1A roles in mRNA translation we identified small molecules affecting eIF1A-RPS10 interaction and use this molecules to affect this interaction in fixed cells using proximity ligation assay.

Imaging Methods: confocal microscopy

Organisms: Homo sapiens

Drosophila male germline stem cells (GSCs) reside at the testis tip, surrounding a cluster of niche cells known as the hub. Bone Morphogenetic Protein (BMP) ligands secreted from the hub exert both contact-dependent and -independent effects. In close proximity to the niche, BMP signaling maintains stem cells by suppressing transcription of the key differentiation factor Bag of Marbles (Bam). In contrast, the diffusible fraction of BMP promotes differentiation of cells by activating bam. How a single signaling pathway produces such opposing outcomes has remained unclear. Here, we show that the diffusible BMP fraction induces bam transcription by repressing the transcriptional repressor Brinker (Brk). We further found that brk mRNA displays a highly heterogeneous expression pattern within interconnected spermatogonia, suggesting that Brk may prime cell fate in a subset of transit-amplifying cells, helping to preserve a population poised for dedifferentiation while maintaining other cells for differentiation. Our findings propose a model in which a single niche-derived factor modulates reciprocal outcomes inside versus outside the niche, which is essential for the tissue homeostasis. Given the broad use of BMP signaling across stem cell niches, this mechanism may represent a general strategy to ensure correct balance between self-renewal and differentiation of stem cells.

Imaging Methods: confocal microscopy

Organisms: Drosophila melanogaster