Nia, Hadi T.Jones, Mitchell2025-05-292025https://hdl.handle.net/2144/505012025Understanding how mechanical and pathological forces interact with the 3D lung structure, cellular populations, and molecular architecture on an organ-wide scale remains a central challenge in pulmonary biology and disease research. Current techniques used to spatially interrogate biology — including histology, medical imaging, and spatial transcriptomics — lack the ability to resolve full 3D organ architecture while simultaneously capturing cellular and molecular information. Additionally, live organ imaging methods such as intravital windows and, in our lab, the Crystal Ribcage have enabled impressive visualization of dynamic processes on the lung surface while preserving physiological function, but visuals are restricted to superficial layers due to light scattering. Emerging techniques using chemical optical clearing have overcome this limitation by matching tissue refractive index and removing light-scattering components in fixed tissues, enabling deep optical imaging across intact organs. Additionally, novel cyclic immunofluorescent multiplexing methods have enabled imaging of many molecular targets within a single sample, though labeling thick tissue sections remains a key challenge. Utilizing these advances, this thesis aims to develop a pipeline for volumetric imaging of the murine lung by integrating serial tissue sectioning, 2,2-thiodiethanol (TDE) optical clearing, and high-resolution confocal microscopy. We focused on (1) optimizing sample preparation, clearing, and imaging for thick, well-oriented lung sections; and (2) aligning slice images to form a complete volume. In part this required registering fluorescent images to a structural scan acquired via section tomography combined with optical coherence tomography, allowing correction of slice deformation and enabling reconstruction of anatomically accurate volumes. This work demonstrates the desired image resolution and alignment for building a functional lung atlas — an integrated 3D map of structural, cellular, and molecular features across the murine lung. Future work will focus on scaling up computation and optimizing multiplex labeling.en-USAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/Biomedical engineeringThesis/Dissertation2025-05-280009-0004-1055-6325