Single-objective light-sheet microscopy enables multiscale imaging spanning from subcellular structures to whole organs

Light-sheet fluorescence microscopy (LSFM) is an advanced three-dimensional microscopic imaging technique, possessing unique imaging advantages for samples of various scales. One the one hand, for mesoscale samples, its tomographic sheet-like illumination allows the acquisition of information from a whole plane at a time, greatly increasing the imaging throughput (the amount of information obtained per unit time). On the other hand, for nanoscale samples, its three-dimensional resolution and photobleaching performance are both superior to those of traditional widefield illumination optical microscopes. However, traditional LSFM's dual-objective setup limits mounting options and speed. Single-objective LSFM (OPM) addresses this, but still has drawbacks like limited field of view and need for additional objectives. Addressing these limitations is crucial for LSFM's full potential in biomedical research. Herein, we propose double-beam interference Microscopy, a novel single-objective LSFM design which could realize horizontal plane illumination using two oblique laser sheets, compatible with various sample mounts. It eliminates remote correction, enabling imaging from organelles to mouse brains by switching objectives. Its sharp optical sectioning and full-aperture detection offer superior spatiotemporal resolution. We showcase its versatility in neuroanatomy and immunology imaging, enabling coarse-to-fine imaging, automatic coordinate mapping, and anatomical annotation correlation.