Traditional histological methods provide limited insights into the complex 3D microstructure of fascia and its relationship to disease. This study explored the capacity of different 3D microscopy techniques for characterizing the microstructure of fascia lata (FL) and thoracolumbar fascia (TLF). Tissues from four donors were studied using optical projection tomography (OPT), digital light sheet (DLS) microscopy, and magnetic resonance microscopy (MRM). Samples for OPT and DLS were imaged with a custom OPT scanner and the DLS arm of a Leica Stellaris microscope, respectively. MRM was performed using a 9.4 T superconducting magnet and an NMR/MRI spectrometer. Reference histological evaluation was performed to guide the interpretation of 3D data. Image analyses were performed using FIJI and Ellipse software. DLS offered superior resolution, but all techniques revealed a trilaminar structure in both fasciae: a thick, collagen-rich intermediate layer flanked by thinner layers with loose connective tissue. The FL intermediate layer was thinner (210.5-258.7 μm) with longitudinally oriented collagen, while the TLF intermediate layer was thicker (302.3-343.6 μm) with both oblique and longitudinal fibers. The superficial layer in FL was thicker (128.8-161.5 μm) than in TLF (84.65-123.10 μm) across imaging modalities. The deep layer also varied between fasciae, with 54.3-73.8 μm in FL and 44.78-70.30 μm in TLF. Layer thickness measurements did not differ significantly across techniques. This study demonstrates the feasibility of different 3D microscopy techniques for visualizing and quantifying fascia extracellular matrix structure and organization, laying the groundwork for future investigations into potential structural alterations in disease.

Department of Condensed Matter Physics Jožef Stefan Institute Ljubljana Slovenia

Institute of Anatomy Faculty of Medicine University of Ljubljana Ljubljana Slovenia

Laboratory of Biomathematics Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

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