Cleared tissue dual-view oblique plane microscopy

We present a dual-view oblique plane microscope (dOPM) for imaging thick optically cleared tissue samples using a silicone immersion primary objective. The custom-designed remote refocusing relay utilizes stock optics to achieve remote refocusing in refractive index-matched samples. The spatial resolution of the system was characterized using a series of fluorescent bead phantoms with refractive indices ranging from 1.4-1.5, with the point spread function full width at half maximum measuring ∼0.5 µm laterally and ∼1 µm axially for a refractive index-matched bead sample, with minimal degradation over a > 250 µm axial range. We characterize how the remote refocusing performance at sample refractive indices up to <i>n</i> = 1.5 can be partially compensated for using adjustment of the correction collar on the primary objective. We apply the system to imaging a range of biological samples with varied refractive indices. Combined with tiled acquisition, image stitching, and multi-view image fusion, the microscope enables multicolour imaging of mm-wide and ∼250 µm thick optically cleared mouse ovarian cancer, bone marrow, and colon tissue samples, and a whole <i>Drosophila melanogaster</i> fruit fly brain. The system offers a platform for fast and high-resolution, multicolour volumetric imaging across spatial scales, integrated on a commercially available microscope frame.