Light-sheet microscopy offers significant advantages for imaging large, live samples due to its speed, gentle illumination, and ability to achieve high resolution. However, conventional light-sheet microscopes often require complex setups and sample preparation. Single-objective light-sheet microscopy aims to simplify this process by using a single objective for both illumination and detection. While this approach simplifies sample handling, it has historically presented challenges in achieving both high resolution and large fields of view. Existing single-objective methods, utilizing oblique plane illumination (OPM), have typically been limited to small fields of view, necessitating time-consuming tiled acquisition. Furthermore, imaging large, complex samples can lead to image quality degradation due to light scattering and absorption. Multi-view imaging, while effective in mitigating these issues, often involves complicated multi-objective setups or sample rotation. DaXi addresses these limitations by incorporating a novel custom remote focusing objective to achieve a wider field of view and high resolution, implementing a fast volumetric scanning modality (Light-sheet Stabilized Scanning, LS³), enabling multi-view imaging with dual oblique illumination, simplifying sample mounting through remote coverslip placement, and demonstrating capabilities via imaging various biological systems.

The development of light-sheet microscopy has revolutionized live sample imaging. Early implementations, like those described by Huisken et al. (2004) and Keller et al. (2008), demonstrated the power of this technique for visualizing developmental processes in zebrafish. Subsequent advancements aimed to improve resolution and imaging speed (McDole et al., 2018; Shah et al., 2019). Single-objective designs, such as those described by McGorty et al. (2015) and Strnad et al. (2016), offered simplified sample handling, but often compromised resolution or imaging speed. Oblique plane microscopy (OPM), initially proposed by Dunsby (2008), showed promise for high-resolution single-objective imaging, particularly with the use of refractive index-mismatched remote focusing (Millett-Sikking & York, 2019; Sapoznik et al., 2020). However, these techniques were often limited in their field of view. Multi-view imaging (Tomer et al., 2012; Krzic et al., 2012; Preibisch et al., 2014; Royer et al., 2016) has emerged as a crucial technique to address issues of light scattering and absorption in large samples. However, these methods often require complex multi-objective setups.

DaXi utilizes a novel optical design based on oblique plane illumination (OPM). A custom tertiary objective (AMS-AGY v.2.0) with a numerical aperture (NA) of 1.0 is employed to achieve high resolution and a large field of view. This objective is crucial in maintaining high image quality over the extended field of view, a challenge addressed by the use of air-glass imaging boundary and zero working distance. The system uses a primary objective (Olympus XLUMPLFLN 20XW) for both illumination and fluorescence collection. Fluorescence is relayed to a secondary objective (Olympus UPLXAPO20X) via a series of tube lenses. A key innovation is the development of Light-sheet Stabilized Scanning (LS³), which combines continuous stage movement with galvo mirror-induced compensatory motion of the light sheet. This allows for large volumetric imaging without motion blur. Multi-view imaging is achieved by employing dual light-sheet illumination and a unique image flipping module, allowing the acquisition of two orthogonal views without significant light loss. The microscope can be easily configured for either upright or inverted imaging via remote coverslip placement. Detailed information on the optical setup, light-sheet generation, scanning methods, and image processing pipeline are provided in the supplementary materials. The system is controlled using Micro-Manager software with custom Python scripts to coordinate hardware components and data acquisition. Post-processing involves steps like deskewing, dehazing, registration, fusion, and temporal stabilization using the open-source Python package ‘dexp’.

DaXi achieves a lateral resolution of approximately 450 nm and an axial resolution of 2 µm. The imaging volume spans 3,000 µm (x) × 800 µm (y) × 300 µm (z), with the x-dimension limited only by the stage travel range (75 mm). The system successfully imaged various biological samples, including whole zebrafish larvae (up to 30 hpf), Drosophila egg chambers, and up to nine zebrafish embryos simultaneously. The multi-view imaging capability significantly improved image quality and coverage. High-speed volumetric imaging was demonstrated, for example, imaging zebrafish whole-brain activity at 3.3 Hz. Time-lapse imaging of zebrafish tail development over several hours was also achieved, allowing observation of cellular processes such as somitogenesis and cell division at high spatio-temporal resolution. Quantitative analyses of the point spread function (PSF) were conducted and found to be consistent across the imaging volume and for different color channels. The instrument's throughput is at least 2.6 times higher than previous similar systems. The remote coverslip method enables easy switching between upright and inverted microscopy configurations, increasing the versatility of the system.

DaXi represents a significant advancement in single-objective light-sheet microscopy. The combination of high resolution, large imaging volume, multi-view capability, and high speed addresses many of the limitations of previous systems. The LS³ scanning method effectively eliminates motion blur, allowing for the acquisition of high-quality images of large samples. The ability to image multiple samples simultaneously significantly increases throughput and enables high-throughput screening applications. The results demonstrate the instrument's applicability for various developmental biology studies requiring long-term, high-resolution imaging of entire organisms. The ease of sample mounting and the flexibility of the system design make DaXi accessible to a broad range of researchers.

DaXi offers a powerful new tool for high-resolution, large-volume imaging of biological samples. Its innovative design, combining a custom high-NA objective, LS³ scanning, dual-view illumination, and remote coverslip placement, overcomes many limitations of previous single-objective light-sheet microscopes. The demonstrated capabilities highlight its potential for a wide range of biological applications, including developmental biology, neuroscience, and high-throughput screening. Future work could focus on incorporating adaptive optics to further improve resolution and explore the integration of additional modalities, such as microfluidics, for even more sophisticated experiments.

While DaXi offers significant improvements, some limitations exist. The current system's axial resolution could potentially be enhanced further. The optical aberrations, primarily primary spherical aberration, though partially compensated, could be further minimized using adaptive optics. The image fusion algorithm, although effective, could be refined for further improvements in image quality. The scalability of the multi-well imaging approach, while demonstrated with nine embryos, could be further explored to optimize for even higher throughput.