A Novel Approach for the Colour Deconvolution of Multiple Histological Stains

Colour Deconvolution (CD) is a commonly used tool in histological medical image analysis that separates histochemical or immunohistochemical stains into their component parts. Traditional CD uses matrix inversion to change the Red, Green and Blue (RGB) channels of an image into a new domain that is representative of reference colours but this limits the total number of stains that can be separated. This is problematic for histological staining protocols that use more than three stains, such as FAST staining. This limitation has restricted the use of multicolour staining in light microscopy. To address this issue, this paper evaluates the use of Non Negative Matrix Factorisation (NNMF) and Non Negative Least Squares (NNLS) to enable the decomposition of multistained histological sections into its source components. It will be shown that NNLS is better suited to imaging modalities such as Whole Slide Image (WSI) scanners and that the multiple staining metrics produced from a single sample are analogous to those generated by applying single reagents to contiguous tissue sections

Experimental and Computational Comparison of Intervertebral Disc Bulge for Specimen-Specific Model Evaluation Based on Imaging

Finite element modelling of the spinal unit is a promising preclinical tool to assess the biomechanical outcome of emerging interventions. Currently, most models are calibrated and validated against range of motion and rarely directly against soft-tissue deformation. The aim of this contribution was to develop an in vitro methodology to measure disc bulge and assess the ability of different specimen-specific modelling approaches to predict disc bulge. Bovine bone-disc-bone sections (N = 6) were prepared with 40 glass markers on the intervertebral disc surface. These were initially magnetic resonance (MR)-imaged and then sequentially imaged using peripheral-qCT under axial compression of 1 mm increments. Specimen-specific finite-element models were developed from the CT data, using three different methods to represent the nucleus pulposus geometry with and without complementary use of the MR images. Both calibrated specimen-specific and averaged compressive material properties for the disc tissues were investigated. A successful methodology was developed to quantify the disc bulge in vitro, enabling observation of surface displacement on qCT. From the finite element model results, no clear advantage was found in using geometrical information from the MR images in terms of the models’ ability to predict stiffness or disc bulge for bovine intervertebral disc.

An In Vitro Study of the Intervertebral Disc Structure Using 3 T Magnetic Resonance Imaging

Study Design. An in vitro magnetic resonance imaging (MRI) study. Objective. Investigate the potential of high-field MRI for producing higher quality images of the intervertebral disc (IVD) to better distinguish structural details. Summary of Background Data. Higher spatial and contrast resolution are important advantages when imaging the complex tissue structures in the spine such as the IVDs. However, at present it is challenging to capture the substructural details in the IVD such as the lamellae. Methods. Three MRI sequences; two-dimensional proton-density-weighted Turbo-Spin-Echo (PD-TSE), 2D T2-weighted Turbo-Spin-Echo (T2W-TSE) with fat-saturation (FS), and 3D Spoiled-Gradient-Echo (3D-GE), were modified based on the image quality and scan duration. IVDs of three intact cadaveric lumbar-spines (T12–S1, Age 83–94 yr) were imaged using these optimized sequences. Thereafter each IVD was transversely sectioned and the exposed surfaces were photographed. Landmark observations from corresponding MRI slices and photographs were compared to confirm the MRI captured morphology. The image quality was evaluated using signal-to-noise ratio (SNR), and relative-contrast values. Finally, the underlying tissue structures, including specific pathological features, were qualitatively compared between the MR images and photographs. Results. Observations from photographs and corresponding MRI slices matched well. The PD-TSE sequence had better overall SNR, but the relative contrast between the tissue types was relatively poor. The 3D-GE sequence had higher relative contrast between the IVD and bone, but not between annulus and nucleus regions. The T2W images provided the best relative contrast between the annulus and nucleus, however the standard deviations here were high. Structural details including fissures, vascular and granular tissue proliferation, and pathologies in the endplate region, were identifiable from the MR images obtained using the optimized sequences. Conclusion. The results demonstrate the potential of high-field MRI to capture the IVD structural details. Since the acquisition durations were within clinically acceptable levels, these methodological improvements have the potential to enhance clinical diagnostics

A cohesive interface approach to model the inter-lamellar behaviour of the intervertebral disc annulus fibrosus

INTRODUCTION - The method of representing the inter-lamellar behaviour in Finite Element (FE) models of the disc has been shown to affect the gross disc mechanics [1]. This study aim was to develop an approach to produce inter-lamellar models of the Annulus Fibrosus, i.e. constitutive models of interaction between lamellae. METHODS (Figure 1) - Mature ovine intervertebral discs were used in the present study as a model for the discs of the human lumbar spine. The annulus fibrosus was dissected and radial slices were subjected to mechanical tests. Images were taken under conventional and differential interference contrast (DIC) microscopy. The images before and during loading were used to produce 2D computational models of the annulus structural behaviour. The DIC images were analysed (ImageJ, U.S. N.I.H., USA) to extract the edges between lamellae and converted into quadrilateral FE meshes (ScanIP 5.1, Simpleware Ltd, UK, and Abaqus 6.12, Dassault Systèmes). Each lamella was modelled with Holzapfel’s anisotropic hyperelastic constitutive behaviour, with one fibre orientation per lamella. For this, model parameters were identified to fit to experimental data on the behaviour of tissue components (the proteoglycan matrix and the collagen fibres) [1]. Several modelling hypotheses were tested for the inter-lamellar behaviour: fully bonded conditions, simple frictionless behaviour, friction behaviour with a Coulomb contact, and delamination behaviour, with or without friction, through the use of a cohesive interface model. Displacement boundary conditions were applied reproducing the measured external displacement on the conventional microscopy images. Adequacy of the inter-lamellar behaviour model was assessed by comparing computational and experimental deformed geometries, specifically the change in lamellar interfaces

A non-destructive method to distinguish the internal constituent architecture of the intervertebral discs using 9.4 Tesla Magnetic Resonance Imaging

Study Design: An in-vitro study of the intervertebral disc (IVD) structure using 9.4T Magnetic Resonance Imaging (MRI). Objective: Investigate the potential of ultra-high-field strength MRI for higher quality 3D volumetric MRI data-sets of the IVD to better distinguish structural details. Summary of Background Data: MRI has the advantages of being non-destructive and three dimensional in comparison to most techniques used to obtain the structural details of biological tissues, however its poor image quality at higher resolution is a limiting factor. Ultra-high-field MRI could improve the imaging of biological tissues but the current understanding of its application for spinal tissue is limited. Methods: Two ovine spinal segments (C7T1, T2T3) containing the IVD were separately imaged using two sequences; 3D-Spin-Echo (multislice-multiecho) pulse sequence for the C7T1 sample and 3D-Gradient-Echo (Fast-Low-Angle-Shot) pulse sequence for the T2T3 sample. The C7T1 sample was subsequently decalcified and imaged again using the same scanning parameters. Histological sections obtained from the decalcified sample were stained followed by digital scanning. Observations from corresponding MRI slices and histological sections were compared as a method of confirmation of morphology captured under MRI. The signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and relative-contrast values were calculated for quantitative evaluation of image quality. Results: Measurements from histology sections and corresponding MRI slices matched well. Both sequences revealed finer details of the IVD structure. Under the spin-echo sequence, the annulus lamellae architecture was distinguishable and the SNR and CNR values were higher. The relative contrast was considerably higher between high (nucleus) and low (bone) signal constituents, but between the nucleus and the annulus the relative contrast was low. Under the gradient echo sequence, although the relative contrasts between constituents were poor, the fibre orientation was clearly manifested. Conclusions: The obtained positive results demonstrate the potential of ultra-high-field strength MRI to non-destructively capture the IVD structure

Structural modelling of the annulus fibrosus - an anisotropic hyperelastic model approach at the lamellar level

This work was aimed at developing a first approach to produce structural models of the Annulus Fibrosus, i.e. constitutive models of individual lamellae. Parameters of Holzapfel’s model were fitted using literature data on the behaviour of tissue components. Used on sample-specific or simplified geometries, the effect on the whole annulus deformation of the fibre angles, interlamellar behaviour, and incompressibility was analysed to assess their role in the disc mechanics