An Optimized Freeze‐Dry Multimodal Workflow for Sequential Micro‐CT Imaging, Histology, and Molecular Profiling: A Use Case in Human Liver Fibrosis

Abstract

ABSTRACT 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.