A quality optimization approach to image Achilles tendon microstructure by phase-contrast enhanced synchrotron micro-tomography

Abstract

Achilles tendons are mechanosensitive, and their complex hierarchical structure is in part the result of the mechanical stimulation conveyed by the muscles. To fully understand how their microstructure responds to mechanical loading a non-invasive approach for 3D high resolution imaging suitable for soft tissue is required. Here we propose a protocol that can capture the complex 3D organization of the Achilles tendon microstructure, using phase-contrast enhanced synchrotron micro-tomography (SR-PhC-μCT). We investigate the effects that sample preparation and imaging conditions have on the resulting image quality, by considering four types of sample preparations and two imaging setups (sub-micrometric and micrometric final pixel sizes). The image quality is assessed using four quantitative parameters. The results show that for studying tendon collagen fibers, conventional invasive sample preparations such as fixation and embedding are not necessary or advantageous. Instead, fresh frozen samples result in high-quality images that capture the complex 3D organization of tendon fibers in conditions as close as possible to natural. The comprehensive nature of this innovative study by SR-PhC-μCT breaks ground for future studies of soft complex biological tissue in 3D with high resolution in close to natural conditions, which could be further used for in situ characterization of how soft tissue responds to mechanical stimuli on a microscopic level.

Significance of the broad non-bony attachments of the anterior cruciate ligament on the tibial side

“…Finally, this study was purely anatomical; therefore, we could not provide quantitative measurements related to ACL force. Techniques, such as contrast-enhanced X-ray CT and phase contrast-enhanced synchrotron X-ray micro-tomography, may be useful for the quantitative evaluation of the ACL fascicle alignment 45 – 47 . In the future, a biomechanical study may provide additional information regarding the mechanisms of ACL injury.…”

Phase-contrast enhanced synchrotron micro-tomography of human meniscus tissue

“…The thawed meniscus samples were imaged using SR-PhC-mCT at the TOMCAT beamline at the Swiss Light Source (Paul Scherrer Institute, Switzerland) 22 . The setup was optimized as previously described for tendon tissue by Pierantoni et al 13 . The images were acquired using the High Numerical Aperture Microscope setup (4 magnification, field of view of 4.2 mm  3.5 mm and pixel size of 1.625 mm) coupled to a LuAG:Ce scintillator screen of 150 mm.…”

“…The power spectrum was calculated by applying a fast Fourier transform to the data and spatial resolution was defined as the frequency where the intensity was two times the noise level, as suggested by Modregger et al 25 . The noise level was defined as the value that the power spectrum converged to which was found by averaging the spectrum over the last third of the frequencies 13,25,26 . Spatial resolution can be defined as the minimum distance at which two features can be resolved in the image 27 .…”

“…Synchrotron radiation-based phase contrast enhanced micro computed tomography (SR-PhC-mCT) has the potential of resolving individual fibers and fiber bundles and to directly visualize collagen organization in a 3D volume. This method also offers the possibility to image tissue samples without fixation or other preparation of the tissue 13 and may therefore be suitable for studies of meniscus biomechanics. SR-PhC-mCT has been performed with promising results in soft musculoskeletal tissues, e.g., intravertebral discs, articular cartilage, and tendon 10,11,13 .…”

“…This method also offers the possibility to image tissue samples without fixation or other preparation of the tissue 13 and may therefore be suitable for studies of meniscus biomechanics. SR-PhC-mCT has been performed with promising results in soft musculoskeletal tissues, e.g., intravertebral discs, articular cartilage, and tendon 10,11,13 . Horng et al also performed CT with SR-PhC of entire human knee joints 9 .…”