![]() DaXi achieves this goal by means of several innovations (Fig. Da and Xi are both words from Chinese Mandarin, where Da refers to the large imaging volume and Xi for the high-resolution capability. To address both of these problems, we present DaXi: a new single-objective design that is capable of imaging large samples, with a large imaging volume, uncompromised image resolution and speed, and multi-view imaging. However, multi-view imaging often requires either complex multi-objective configurations that complicate sample mounting, or sample rotation that decreases imaging speed. An effective solution is multi-view imaging, where samples are illuminated and detected from different orientations and images with better coverage and overall quality can be computationally reconstructed 1, 16, 17, 18, 19. The standard solution to this problem is to tile the acquisition over multiple fields of view, sacrificing imaging speed.Īnother limitation of light-sheet microscopy when applied to large living specimens such as zebrafish embryos is the presence of structures in the sample that absorb, refract and scatter both the illumination and detection light. This field of view cannot accommodate large living samples such as developing embryos and fundamentally limits imaging throughput. However, full NA detection imaging has only been demonstrated with high magnification systems (×100 and ×60), for a limited field of view up to 200 μm 15. This has been addressed more recently via refractive index-mismatched remote focusing 13, 14, 15, which unlocks the potential of OPM for high-resolution imaging by giving it access to the full NA for detection. As a drawback, OPM traditionally was unable to use the full numerical aperture (NA) of the primary objective for fluorescence detection, lowering its resolution and sensitivity. This ‘single-objective’ light-sheet microscope configuration is compatible with diverse specimens including the intact rodent brain 11 and multi-well plated samples 12, 13. This configuration trades optical complexity for sample mounting simplicity, that is additional optical elements (including two remote objective lenses) are added on the detection path so that there is only one objective at the proximity of the sample. The fluorescence is first relayed to a secondary objective, then relayed to a tertiary objective and finally focused onto a camera. Among these methods, the oblique plane microscope (OPM) 10 uses a single-objective lens (referred to as the primary objective) for both illumination and detection, without additional reflecting elements at the sample space. Recent developments have attempted to improve the sample mounting ergonomics with open-top imaging systems 5, 6, 7, 8 or by alleviating the necessity for orthogonal objectives 9. However, a major limitation in light-sheet microscopy is the requirement of complex multi-objective configurations that complicate sample mounting. ![]() The ability to image whole developmental arcs and to follow hundreds of thousands of cells in space and time has shown the potential of light-sheet microscopy in answering long-standing questions 3, 4. It has had a particular impact on developmental biology, allowing the first in toto volumetric reconstructions of embryonic development of model organisms such as Drosophila, zebrafish and mouse 1, 2, 3. In recent years, light-sheet microscopy has become an essential imaging method for biology 1, 2. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development – up to nine embryos at a time. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes.
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