Fig. 1.

Multi-immersion open-top light-sheet (OTLS) microscope. a The system enables simple mounting of multiple specimens with modular transparent holders. Illumination and collection objectives are located underneath the specimen holders and are separated by a liquid reservoir filled with an interchangeable immersion medium. b The off-axis illumination light sheet and collected fluorescence travel obliquely through the immersion media, holder, and specimen. Aberrations are minimized by precisely matching the refractive index of all three materials, and by utilizing the wavefont-matching properties of a solid-immersion lens (SIL) along the illumination path. The depth-of-focus and beam waist of the light sheet are depicted in the inset (upper right). The point spread function (PSF) of the system (scale bars: 1 μm) and refractive-index-dependent numerical aperture (NA) of the illumination and collection beams are shown in (c) and (d). e The mechanical design of the system includes a motorized XY stage, motorized Z actuators, motorized filter wheel, scanning mirror, computer-controlled multi-wavelength fiber-coupled laser package, and sCMOS camera, all of which enable high-throughput automated imaging of multiple specimens simply placed on a flat plate, or placed within a diverse assortment of transparent holder designs (f). g Volumetric imaging is achieved by using a combination of stage scanning and lateral/vertical tiling