Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

Abstract

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.

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

Fig. 2

Holder design for OTLS imaging. a Optical simulations of the system’s point spread function (PSF) and Strehl ratio (S), and ray-tracing spot diagrams, are shown for scenarios in which the optical path difference (Δn × t) is negligible, small, or large, which results in diffraction-limited (S ~ 1.0), near-diffraction-limited (S > 0.8), or aberrated (S < 0.8) imaging performance, respectively (scale bars: 1 μm). The dependence of the Strehl Ratio, S, as a function of Δn × t is plotted, indicating that for diffraction-limited imaging, the condition that Δn × t < 0.002 mm should be maintained. Based on this condition, potential glass and monomer/polymer holder materials are shown in (b). The color scale indicates the maximum material thickness, t_max, that is allowed based upon the intrinsic mismatch, Δn, of those materials with published clearing protocols. Chemically incompatible combinations of materials and chemical reagents are also indicated

Fig. 3

Multi-immersion and multi-specimen imaging. a OTLS imaging of 12 ECi-cleared human prostate biopsies (scale bar: 1 mm) placed within a multi-biopsy holder. Zoomed-in views illustrate the complex 3D structure of benign and malignant glands (scale bars: 25μm and 10 μm). Images can be false-colored to mimic the appearance of conventional chromogen-based (absorption-based) H&E and IHC histopathology. b High-throughput imaging of eight TDE-cleared mouse brain slices placed on a 10 × 10 -cm glass plate (scale bar: 1 mm). A higher-magnification region of interest demonstrates the ability to visualize individual neurons (scale bar: 100 μm). c Whole-organ imaging of Ce3D-cleared mouse lymph nodes, heart, prostate, and lung placed within an multi-well plate (scale bars: 1 mm). d Large-scale imaging of an expanded thick-kidney slice placed on a teflon drumhead. High-resolution regions of interest show individual glomeruli (scale bar: 40 μm), vessels (scale bar: 80 μm), and tubules (scale bar: 50 μm). A multi-channel zoom-in with DAPI counterstaining demonstrates the increased resolution due to expansion (scale bars: 100μm and 20 μm). The scale bars for the entire expanded kidney indicates the physical size of the expanded specimen, whereas the scale bars for the insets indicate the dimensions of the native unexpanded tissue