Contributors: Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology; Institut Pasteur Paris (IP)-Université Paris Cité (UPCité); Princeton University; Department of Physics, Princeton University (DPPU); This work was supported by Institut Pasteur, Centre National de la Recherche Scientifique, grants from Region Ile-de-France (DIM ELICIT), the French National Research Agency (ANR-20-CE12-0028’ChroDynE’ and ANR-23-CE13-0021’GastruCyp’ and ANR-10 LABX-73’Revive’), and by funding from the European Research Council (ERC-2023-SyG, Dynatrans, 101118866). LF is also supported by CFM Foundation for Research; ANR-20-CE12-0028,ChroDynE,Dynamique de la chromatine et transcription durant la mitose dans les cellules souches mammifères(2020); ANR-23-CE13-0021,GastruCyp,Régulation transcriptionnelle de Cyp26a1 pendant l'extension de l'axe chez des gastruloïdes mammifères(2023); ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010); European Project: 101118866,ERC-2023-SyG,ERC-2023-SyG,DynaTrans(2024)
نبذة مختصرة : Quantitative imaging of subcellular processes in living embryos, stem-cell systems, and organoid models requires microscopy platforms that combine high spatial resolution, fast volumetric acquisition, long-term stability, and minimal phototoxicity. Single-objective light-sheet approaches based on oblique plane microscopy (OPM) are well suited for live imaging in standard sample geometries, but most existing implementations lack the optical calibration, timing precision, and end-to-end integration required for reproducible quantitative measurements.Here we present a fully integrated and quantitatively characterized OPM platform engineered for dynamic studies of transcription and nuclear organization in embryos, embryonic stem cells, and threedimensional culture systems. The system combines high-numerical-aperture remote refocusing with tilt-invariant light-sheet scanning and hardware-timed synchronization of laser excitation, galvo scanning, and camera readout. We provide a comprehensive characterization of the optical performance, including point-spread function, sampling geometry, usable field of view, and system stability, establishing a well-defined framework for quantitative volumetric imaging. To support high-throughput operation, we developed a unified acquisition and reconstruction pipeline that enables real-time volumetric imaging at hardware-limited rates while preserving deterministic timing and reproducible geometry. Using this platform, we demonstrate quantitative threedimensional imaging of MS2-labeled transcription sites in living Drosophila embryos, cultured mouse embryonic stem cells, and mESC-derived gastruloids, enabling extraction of transcriptional intensity traces across diverse biological contexts.Together, this work establishes OPM as a robust and quantitatively calibrated single-objective light-sheet platform for transcription imaging in complex living systems, providing a foundation for future studies of gene regulation and nuclear dynamics across developmental and stem-cell models
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