1.2 Biological materials
2.1.2 Experimental procedure
For studying the microscopic displacements of entheses under load, the trade off between image size and image acquisition time needs to be addressed, while bearing in mind that the aim is to investigate the tendon-bone insertion as a whole, functional biomaterial. The preferable approach is one that allows to directly relate micromechanical responses with macroscopic ones; for this reason images that cover the whole sample with high resolution are used. The areas to be imaged are 1 cm2to 2 cm2, typically 5 mm×8 mm. Additionally, a z-thickness of the sample is also sampled via stacks of confocal slices, to account for the ruggedness of the sample surface and to capture any largez-displacement that might ensue as a byproduct of the axial testing. Typically the necessary z-scanning is 300µm to 500µm.
Besides these spatial requirements, the timing and duration of the experi-ments needs to be also taken into account. The total duration of the experiment must not be so long as to pose a serious concern for the integrity of the biological samples. Long term exposure to various buffers has been shown to impact the structure of tendon fibers on the micrometre scale, by causing swelling. Even buffers that are broadly regarded as physiological, such as phosphate buffered saline (PBS) and Dulbecco’s modified eagle medium (DMEM), have been shown to have this effect[86]. As a general guideline, the experiments should be as fast as possible, to avoid any sample degradation of this or any other kind.
To comply as best as possible with these diverse requirements the imaging was performed with a 10×objective (NA=0.3, Leica PL Fluotar) with a field of view of 1550µm×1550µm, to obtain images 512 px×512 px with 3.027µm px−1. This is not the optimal pixel size to exploit the best resolution achievable by the objective but, as will be discussed in 2.3.3, it is enough to study displacements on the 100 nm scale. To cover the whole sample several images were acquired as tiles of a grid, with 20 % overlap between neighboring tiles. Each tile was itself az-stack of approximately 100 slices with 3µm spacing between slices.
Scanning was performed as fast as possible, at 500 line s−1, resulting in a typical time for a full grid of stacks to be acquired of 40 min.
The experimental procedure consists of the application of increasing levels of strain, following a series of incremental steps. Grids of stacks are acquired
between two strain increments, while the sample is being maintained at a constant strain. Under these conditions, the viscoelastic behavior of tendon causes a stress-relaxation effect, therefore the sample is allowed to relax for a timeτrelaxbefore acquiring images after each application of strain. From stress relaxation curves a decay time of∼20 s was extracted, and the value ofτrelax
was set to 2 min.
The complete duration of an experiment depends on how many strain in-crements are applied and imaged, which in total were between 5 and 10. Each strain step was applied as a deformation 0.5 mm, such a step corresponded to approximately 1 % strain. The readings of the force sensor resulting from the applied deformation were recorded to plot stress-strain curves for each sample.
From these curves the regime of linear response of the each sample was identified (see appendix B).
Strain measurement The experiments have been described up to now as applying stepwise strain increments to a sample. The loading chamber allows for control of the applied deformation via the motor, and the strain can be calculated from the deformation according to
"=ln(1+"e) ="e−"2e/2+"3e/3+.... (2.1) The so-called engineering strain "e is given by "e = ∆`/` where ∆` is the elongation along a given axis and ` is the original length along that same axis. In practice, because only small strains are applied, the nonlinear terms in equation 2.1 can be neglected. Thus, to calculate the applied strain, the original length of the undeformed tendon needs to be measured. This was easily done by taking a picture of the samples in the load chamber using a digital camera (such as the inset in fig. 2.1). From this picture the average length`of the tendon between bone and clamp can be directly measured.
Imaging In section 1.2.1 the notable optical properties of collagen were briefly touched upon. For the purpose of imaging the enthesis, the high-intensity reflected signal yielded by collageneous structures is greatly useful. The collagen content of tendons, bone and even cartilage can be visualized with high-specificity by simply detecting this reflected signal, through which collagen organization down to the diffraction limit of the optical system can be investigated. This imaging method is usually referred to as Confocal Reflection (or Reflectance) Microscopy (CRM). In combination with CRM, fluorescence labeling can also be used, to specifically visualize non-collageneous components or to distinguish between the families of collagens, which is not possible by reflectance alone.
In fig. 2.2 a scheme of the full experimental procedure is shown. In the bottom two panels, the steps relating to imaging are described, showing the acquisition of a grid of CRMz-stacks. The top right-hand panel shows the stepwise application of strain and the image acquisition timing. The final result is a sequence of
Figure 2.2: Overview of the experimental procedure, showing how full-sample images are acquired during a quasi-static deformation of an enthesis sample.
image grids, each corresponding to a controlled level of constant strain". Post-processing of the data consisted in performing az-projection on each stack and then stitching the tiles together to obtain a single sample-wide image. Both these steps were carried out using the image analysis softwareImageJ, in its bio-imaging oriented distributionFiji[110,111]. The post-processing was performed as follows.
z-projection A custom written ImageJ macro was used to analyze each slice in the stack. If more than half the pixels in a slice were at the saturation value (255 in the case of 8-bit images), the slice was discarded. This was done to remove the slices dominated by reflections coming from the coverslip.
A background removal algorithm was applied to all remaining slices. The stack was then projected to obtain a single image using a simple pixelwise sum.
Stitching The grid ofz−projections was combined into a single image using a dedicated ImageJ plugin[112]. The plugin fuses the tiles with subpixel accuracy using cross-correlations to refine the known overlap between the tiles. The principles of this algorithm are based on the same concepts reported in section 2.3. In fig. 2.3 a stitched CRM image of an enthesis sample is shown.
Figure 2.3: A confocal reflectance picture showing the tendon-bone insertion of a porcine Achilles tendon. In the top half of the image, the calcaneus bone is visible, while the bottom half is the tendon. Running across the width of the image is the concave curve of the interface between these two tissues. The scale bar corresponds to 1000µm.