An integrated quantitative single-objective light-sheet microscope for subcellular dynamics in embryos and cultured multicellular systems

TL;DR

This paper introduces a fully integrated, high-precision single-objective light-sheet microscopy platform optimized for quantitative, long-term 3D imaging of subcellular processes in live embryos and cultured multicellular systems, enabling detailed transcriptional analysis.

Contribution

The authors developed a novel, integrated OPM platform with comprehensive optical calibration and real-time imaging capabilities for quantitative subcellular studies in complex live biological systems.

Findings

Successful 3D imaging of transcription sites in Drosophila embryos

Quantitative analysis of transcriptional dynamics in stem cells and organoids

High stability and reproducibility demonstrated in diverse biological samples

Abstract

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 three-dimensional 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 three-dimensional 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. This work establishes OPM as a robust and quantitatively calibrated single-objective light-sheet platform for transcription imaging in complex living systems.