129 - Comparison of Microscopy Techniques to Capture 3D Native State of Fascia Microarchitecture

Poster Board Number: 129
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Abstract Body : Introduction and Objective: Musculoskeletal (MSK) pain is one of the leading causes of disability worldwide. Research begins to advance the importance of fascia in pathophysiology related to MSK pain, such as the thoracolumbar fascia and low back pain. One of the fascial system’s functions is to equilibrize forces between anatomical structures. Research on connective tissues, like bone, has demonstrated that there is a relationship between the impact of mechanical forces on the reorganization of the tissue’s microarchitecture. Understanding the 3-dimensional (3D) microarchitecture of fascia would help better understand fascia related disorders that could explain MSK pain. Traditional histology preparation methods used to study the microarchitecture of fascia often introduce artifacts and alter the 3D native state of the tissue samples. This study explored tissue sample preparation procedures and several microscopy techniques to best characterize the preserved 3D microarchitecture of thoracolumbar fascia.

Materials and Methods: With IRB approval, embalmed human thoracolumbar fascia samples were obtained through dissection-based isolation. These samples were stored in 4% paraformaldehyde (PFA) solution and refrigerated until use. Four microscopic imaging approaches were selected for image acquisition: Zeiss Widefield Axioscope with polarized light microscopy features; Zeiss LSM710 laser scanning confocal microscope where images and z-stacks were taken; a home-built second harmonic generation (SHG) microscope where z-stacks and tiles were taken; Zeiss Xradia Versa 520 where the entire fascia sample was scanned after critical point drying (CPD) sample preparation. After image acquisition, data was subjected to image analysis using ImageJ to identify any information on the collagen fiber orientations and IMARIS to generate 3D reconstructions.

Results: The Zeiss Widefield Axioscope allowed for the collagen fibers to be easily seen and their orientations to be identified. The polarized images show assorted colors that run in different orientations to represent distinct types of collagens in the tissue sample. However, the results from this imaging method are 2-dimensional and does not allow for visualization of the multiple collagen layers. The Zeiss LSM710 laser scanning confocal microscope revealed some collagen fibers, but the entire depth of the fascia sample could not be imaged. This technique was only capable of imaging up to 15um to 20um into the sample. By using SHG, a much better depth was achieved, up to 100um deep. With SHG, collagen fibers orientations are more easily visualized and quantified. Lastly, using the Xradia Versa 520, microcomputed tomography (micro-CT) scanning was used on CPD-prepared fascia to allow for the entire fascia sample to be imaged and reconstructed in 3D with all layers of collagen easily seen.

Conclusion: We present two methods for visualizing the dense collagen network in thoracolumbar fascia without strong detergents or tissue sectioning. Both second harmonic generation imaging and micro-CT scanning combined with image processing allows for the dense collagen network to be revealed in thick fascia tissue.

Significance/Implication: The ability to image the 3D native state of the collagen network of deep fascia will enable further 3D morphometric analyses of the microarchitecture to characterize better its properties and variation related to specific pathological conditions.