An Optimized Freeze-Dry Multimodal Workflow for Sequential Micro-CT Imaging, Histology, and Molecular Profiling: A...

Classical histology remains the gold standard for liver fibrosis assessment but is limited by destructive sectioning, 2D information, and sampling bias. Micro-CT offers non-destructive 3D imaging, yet conventional contrast agents can compromise tissue integrity and downstream analyses. This study establishes a resource-efficient multimodal workflow that enables high-quality imaging and subsequent histological and molecular testing from the same specimen. FFPE normal and cirrhotic liver tissues (n = 3 each) undergo an optimized micro-CT protocol: DPBS wash, 30% eosin incubation, dehydration, snap-freezing, and lyophilization. After imaging, tissues are rehydrated, re-embedded in paraffin, and processed for routine histology, immunohistochemistry, RNA extraction with qPCR, and targeted NGS (800-gene panel). The protocol yields high-contrast, artifact-free micro-CT images with clear visualization of nuclei, sinusoidal networks, portal structures, and fibrotic septa (∼20x-40x optical equivalent). Volumetrics show minimal fibrosis in normal liver (2.8%) versus extensive remodeling in cirrhosis (43.5%). Morphology and antigenicity are preserved (H&E, PSR, IHC). RNA quality supports qPCR, revealing upregulation of inflammatory and cirrhosis-related genes in cirrhosis. Targeted NGS libraries are successfully generated, with protocol-dependent performance differences. This minimally destructive workflow maximizes information yield from limited tissue, enabling integrated imaging and molecular profiling with broad utility in pathology, biobanking, and translational research.