Quantifying the hydroxyapatite orientation near the ossification front in a piglet femoral condyle using X-ray diffraction tensor tomography

While a detailed knowledge of the hierarchical structure and morphology of the extracellular matrix is considered crucial for understanding the physiological and mechanical properties of bone and cartilage, the orientation of collagen fibres and carbonated hydroxyapatite (HA) crystallites remains a debated topic. Conventional microscopy techniques for orientational imaging require destructive sample sectioning, which both precludes further studies of the intact sample and potentially changes the microstructure. In this work, we use X-ray diffraction tensor tomography to image non-destructively in 3D the HA orientation in a medial femoral condyle of a piglet. By exploiting the anisotropic HA diffraction signal, 3D maps showing systematic local variations of the HA crystallite orientation in the growing subchondral bone and in the adjacent mineralized growth cartilage are obtained. Orientation maps of HA crystallites over a large field of view (~ 3 7 3 7 3 mm3) close to the ossification (bone-growth) front are compared with high-resolution X-ray propagation phase-contrast computed tomography images. The HA crystallites are found to predominantly orient with their crystallite c-axis directed towards the ossification front. Distinct patterns of HA preferred orientation are found in the vicinity of cartilage canals protruding from the subchondral bone. The demonstrated ability of retrieving 3D orientation maps of bone-cartilage structures is expected to give a better understanding of the physiological properties of bones, including their propensity for bone-cartilage diseases

Matlab code for an automated P-SHG setup on a commercial microscope

First release of the matlab code composed to control and calibrate a P-SHG setup designed for commercial microscopes

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes - Fig 4

<p>(a, b) An overview of the ellipticity, <i>ρ</i>, between the minimum and maximum electric field component of the polarization for different HWP and QWP combinations for two different microscopes. For linearly polarized light the ellipticity is 0, and for circularly polarized light the ellipticity is 1. The overlaying plots indicate which waveplate combinations generate linearly polarized light (black lines) and circularly polarized light (black and white circles) as determined by fitting a theoretical model. The white crosses in each plot indicates the HWP and QWP settings used when measuring the polarization illustrated in (c) and (e). The white circles correspond similarly to plots (d) and (f). The single measurements had an ellipticity of <i>ρ</i> = 0.29 (c), <i>ρ</i> = 0.86 (d), <i>ρ</i> = 0.19 (e) and <i>ρ</i> = 0.97 (f).</p

An illustration of the orientation and rotation of the QWP and HWP compared to the polarization of the incoming laser light.

<p>The motors containing the QWP and HWP are not calibrated according to the <i>x</i>-axis. Therefore, a relative angle <i>ϕ</i> = <i>ϕ</i>′ − <i>ϕ</i>₀ and <i>θ</i> = <i>θ</i>′ − <i>θ</i>₀ have to be implemented for the QWP and HWP, respectively. Here <i>ϕ</i>′ and <i>θ</i>′ are the angles provided by the motors, and <i>ϕ</i>₀ and <i>θ</i>₀ are the angles of the fast axis of the QWP and HWP, respectively, compared to the <i>x</i>-axis when the motors are at 0°.</p

Comparison of Compressive Stress-Relaxation Behavior in Osteoarthritic (ICRS Graded) Human Articular Cartilage

Osteoarthritis (OA) is a common joint disorder found mostly in elderly people. The role of mechanical behavior in the progression of OA is complex and remains unclear. The stress-relaxation behavior of human articular cartilage in clinically defined osteoarthritic stages may have importance in diagnosis and prognosis of OA. In this study, we investigated differences in the biomechanical responses among human cartilage of ICRS grades I, II and III using polymer dynamics theory. We collected 24 explants of human articular cartilage (eight each of ICRS grade I, II and III) and acquired stress-relaxation data applying a continuous load on the articular surface of each cartilage explant for 1180 s. We observed a significant decrease in Young’s modulus, stress-relaxation time, and stretching exponent in advanced stages of OA (ICRS grade III). The stretch exponential model speculated that significant loss in hyaluronic acid polymer might be the reason for the loss of proteoglycan in advanced OA. This work encourages further biomechanical modelling of osteoarthritic cartilage utilizing these data as input parameters to enhance the fidelity of computational models aimed at revealing how mechanical behaviors play a role in pathogenesis of OA

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes - Fig 6

<p>Waveplate combinations that provide linear polarization were selected at polarization angle intervals of 10° from 0° to 180° (a, white crosses and plus signs). There are two different combinations that provide the same polarization angle, thereof the two separate sets (crosses vs plus signs). The polarization angle and ellipticity were measured for all waveplate combinations in both sets (b). Tendon was imaged twice using all waveplate combinations (c, average). Two pixels were selected (c, white asterisk and star) to demonstrate the variation of the intensity as a function of polarization angle for the two sets (d). The second order susceptibility tensor ratios and fiber angle were calculated separately for the two sets (plus signs: e, f, g, crosses: h, i, j). The difference between the two was emphasized by taking subtracting the results from one of the sets (crosses) from the other (plus signs) (k, l, m).</p

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes - Fig 5

<p>(a, b) A detailed and coarser map of the ellipticity, <i>ρ</i>, with overlaying waveplate combinations that provide linearly (mean: black lines, ±3std: dashed lines) and circularly (mean: black circles, ±3std: intervals) polarized light. To illustrate the accuracy smaller regions of interest (a, white rectangles) were measured in more detail (c, d, e). The overlapping plots are based on the fitting of the original map (a), and have an ellipticity of <i>ρ</i> = 0.29 (c), <i>ρ</i> = 0.12 (d) and <i>ρ</i> = 0.86 (e).</p

Characterization of Cholesterol Crystals in Atherosclerotic Plaques Using Stimulated Raman Scattering and Second-Harmonic Generation Microscopy

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue

Media 3: Nonlinear optical microscopy of early stage (ICRS Grade-I) osteoarthritic human cartilage

Originally published in Biomedical Optics Express on 01 May 2015 (boe-6-5-1895