Combined orthogonal and non-orthogonal light-sheet microscopy for omniscale 3D imaging

Abstract

In recent years light-sheet microscopy has emerged as a powerful technique for fast volumetric imaging of live specimens [1] . In light-sheet microscopy, a thin of light optically (rather than physically) sections a specimen, such that fluorescence is only excited within one section. By using weakly-focused illumination light, the sheet maintains its sectioning ability over hundreds of microns, enabling high-speed capture of 2D images. And by scanning the specimen (or light sheet) a 3D dataset is quickly generated [2] . In most systems, the illumination and collection objectives are oriented orthogonally, such that the light sheet is co-planar with the focal plane of the collection objective [3] . However, more recently a class of light-sheet microscopes which use a single objective for both illumination and collection have been proposed. These non-orthogonal designs, known as oblique-plane microscopy (OPM), swept-confocally aligned planar excitation (SCAPE), epi-illumination selective-plane illumination microscopy (eSPIM), and scanned oblique-plane illumination microscopy (SOPi), illuminate samples with a light sheet which is imaged in a non-orthogonal fashion, which is rectified at a tilted remote image focus [4] - [8] . While this non-orthogonal arrangement results in slightly worse imaging performance (compared to the orthogonal arrangement), the use of a single objective enables more convenient and less physically restricted imaging geometries. To date, these non-orthogonal light-sheet microscopes have primarily been explored for dynamic imaging of live specimens, and more recently water-based expanded tissues. Here we combine this non-orthogonal architecture with a more traditional orthogonal light-sheet microscope. The non-orthogonal optical path providing micron to sub-micron resolution, and the orthogonal system providing lower magnification mesoscale imaging. Combined, the single system is able to provide omniscale 3D imaging.