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

Maria Pierantoni,Isabella Silva Barreto,Malin Hammerman,Rajmund Mokso,Elin Törnquist,Pernilla Eliasson,Hanna Isaksson,Lissa Verhoeven,Vladimir Novak

doi:10.1038/s41598-021-96589-w

Maria Pierantoni, Isabella Silva Barreto + Show 7 more

Open Access

https://doi.org/10.1038/s41598-021-96589-w

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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.

Highlights

Achilles tendons are mechanosensitive, and their complex hierarchical structure is in part the result of the mechanical stimulation conveyed by the muscles
Kidney from rat was used as a positive control. As this represents one of the first studies in which the tendon microstructure was imaged by phase contrast enhanced synchrotron tomography, we compared the tomographic data to state of the art histology to show that the internal tendon structure presents as expected (Fig. 3)
After concluding that fresh and fixed samples showed similar image quality, we further investigated for these two sample preparations, if an increase in magnification would translate to a significant increase in image quality

Summary

Introduction

Their complex hierarchical structure is in part the result of the mechanical stimulation conveyed by the muscles. 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 full correlation between the mechanical stimuli and the resulting complex 3D organization of the collagen is yet to be elucidated To unravel this relationship, well-established high-resolution 3D imaging techniques, suitable for imaging soft biological tissues, such as the tendon, without affecting the mechanical properties are warranted. Synchrotron X-ray tomography is a non-destructive method for 3D visualization and quantitative analysis of materials[21] It allows to quickly obtain sub-micrometre resolution images, of relatively large volumes (~ 1 to 200 mm[3] depending on the desired magnification), without invasive or time-consuming sample preparation. In this study contrast-to-noise ratio, signal-tonoise ratio, noise power spectrum, spatial resolution and detail visibility were considered[46]

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