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bioRxiv · 10.1101/2025.05.12.653537

Cleared tissue dual-view oblique plane microscopy

Dvinskikh, L.·Sparks, H.·Ennis, D.·Sarnataro, R.·Carreno, D.·Riglar, D. T.·Cunnea, P.·McNeish, I. A.·Dunsby, C.
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Abstract

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 {micro}m laterally and [~]1 {micro}m axially for a refractive index-matched bead sample, with minimal degradation over a >250 {micro}m axial range. We characterize how the remote refocusing performance at sample refractive indices up to n = 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 {micro}m thick optically cleared mouse ovarian cancer and colon tissue samples with subcellular resolution. We also image a whole Drosophila melanogaster 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.

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Dvinskikh, L., Sparks, H., Ennis, D., Sarnataro, R., Carreno, D., Riglar, D. T., Cunnea, P., McNeish, I. A., Dunsby, C.. 2025-05-15. Cleared tissue dual-view oblique plane microscopy. https://doi.org/10.1101/2025.05.12.653537

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