Wide field-of-view volumetric imaging by a mesoscopic scanning oblique plane microscopy with switchable objective lenses.

Abstract

Conventional light sheet fluorescence microscopy (LSFM), or selective plane illumination microscopy (SPIM), enables high-resolution 3D imaging over a large volume by using two orthogonally aligned objective lenses to decouple excitation and emission. The recent development of oblique plane microscopy (OPM) simplifies LSFM design with only one single objective lens, by using off-axis excitation and remote focusing. However, most reports on OPM have a limited microscopic field of view (FOV), typically within 1×1 mm2. Our goal is to overcome the limitation with a new variant of OPM to achieve a mesoscopic FOV. We implemented an optical design of mesoscopic scanning OPM to allow the use of low numerical aperture (NA) objective lenses. The angle of the intermediate image before the remote focusing system was increased by a demagnification under Scheimpflug condition such that the light collecting efficiency in the remote focusing system was significantly improved. A telescope composed of cylindrical lenses was used to correct the distorted image caused by the demagnification design. We characterized the 3D resolutions and imaging volume by imaging fluorescent microspheres, and demonstrated the volumetric imaging on intact whole zebrafish larvae, mouse cortex, and multiple Caenorhabditis elegans (C. elegans). We demonstrate a mesoscopic FOV up to ~6×5×0.6 mm3 volumetric imaging, the largest reported FOV by OPM so far. The angle of the intermediate image plane is independent of the magnification as long as the size of the pupil aperture of the objectives is the same. As a result, the system is highly versatile, allowing simple switching between different objective lenses with low (10×, NA 0.3) and median NA (20×, NA 0.5). Detailed microvasculature in zebrafish larvae, mouse cortex, and neurons in C. elegans are clearly visualized in 3D. The proposed mesoscopic scanning OPM allows using low NA objectives such that centimeter-level FOV volumetric imaging can be achieved. With the extended FOV, simple sample mounting protocol, and the versatility of changeable FOVs/resolutions, our system will be ready for the varieties of applications requiring in vivo volumetric imaging over large length scales.