Late Breaking Abstract - Reversing cleared lung samples for multi-dimensional analysis in therapeutic discovery

Light-sheet fluorescence microscopy (LSFM) is an emerging imaging technique that enables high-resolution 3D imaging of large, intact samples (up to several centimeters) with subcellular resolution. It is especially powerful for visualizing structural changes in disease and for localizing rare cell types identified in single-cell datasets, capabilities beyond current in vivo imaging modalities. LSFM relies on optical clearing to render tissues transparent and on the immersion of samples in hydrophobic solutions for imaging. Since most labeling approaches use water-based solutions, this precludes further labeling after clearing. This is a key bottleneck for co-localizing multiple cell types in 3D tissues, which is essential for identifying populations defined by multiple phenotypic markers (e.g., immune cell subtypes). To address this, we developed a novel workflow to reverse optical clearing, enabling subsequent histological and RNA-based analyses in the same lung sample. Mouse lungs, embryos, and human fibrotic lung samples were cleared and imaged with LSFM, then reverse-cleared and paraffin-embedded for immunofluorescence and in situ RNAscope analysis. Lung markers such as pro-SPC and AQP5 remained detectable, extracellular matrix components (collagens, glycosaminoglycans) were retained, and RNA yields were comparable to standard paraffin-embedded tissue. Finally, we developed a virtual reality-based co-registration workflow to precisely align 2D histological slices within 3D LSFM datasets. This pipeline expands LSFM's utility, enabling comprehensive, multimodal analysis from a single sample, supporting advanced preclinical and clinical studies of intact tissues at scale.

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