20.500.12556/DiRROS-31845 A high-content imaging pipeline to investigate subcytotoxic effects in RTgill-W1 cells High-content screening offers great potential for in vitro toxicology, yet its application in environmental test systems remains limited by the lack of open, standardized workflows and biologically interpretable analysis frameworks. Here, we established and optimized a reproducible pipeline for image-based phenotypic profiling of rainbow trout gill cells (RTgill-W1) exposed to environmental chemicals, using Tebuthiuron as a case study. The workflow integrates multiple organelle dyes and systematically evaluates critical data processing steps (quality control, standardization, outlier removal, concentration–response-guided feature selection, and dimensionality reduction) to identify robust, biologically meaningful end points. Our systematic comparison demonstrated remarkable robustness: even minimal processing detected concentration–response relationships, while optimized standardization and feature selection substantially improved signal clarity. The framework identified 94 morphological features capturing Tebuthiuron’s concentration- and time-dependent cellular responses, with lysosomal redistribution and mitochondrial fragmentation emerging as early stress indicators. Maximum mean discrepancy and Uniform Manifold Approximation and Projection (UMAP) visualizations provided biologically interpretable maps of phenotypic divergence. This proof-of-concept demonstrates that our quality-controlled, transparent HCS pipeline can resolve concentration- and time-dependent subcellular responses in fish cells, establishing a methodological basis for future multichemical phenotypic profiling. high-content screening phenotyping toxicology fishcells concentration−response ecotoxicology visokovsebinsko presejanje fenotipizacija toksikologija ribje celice odziv na koncentracijo ekotoksikologija true false true Angleški jezik Slovenski jezik Neznano 2026-08-11 09:07:40 2026-08-11 09:07:40 2026-08-12 03:44:48 0000-00-00 00:00:00 2026 0 0 Nasl. z nasl. zaslona; Soavtorji: Barbara Jozef, Sven Lukas Mosimann, Marissa Kosnik, Kristin Schirmer, Anže Županič; Opis vira z dne 10. 8. 2026; str. A-O iss. Vol. 2026 0000-00-00 PostprintKoncna VTisku NiDoloceno 0000-00-00 0000-00-00 2026-07-30 576 1520-5851 10.1021/acs.est.5c18316 287444483 https://doi.org/10.1021/acs.est.5c18316 1 587e0876-9553-11f1-af5a-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=49480 RAZ_Tome_Miha_2026.pdf RAZ_Tome_Miha_2026.pdf 1 BED79F42A7E848DC6633BDB281D6CBAB 2b5d017573b5865c8ef3aa65e4a71ad38a9c1ffb12973dedf2c365f5206a3f7c 6ddb1a28-9553-11f1-af5a-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=49481 Nacionalni inštitut za biologijo 0 0 0