Search

Assisted flipping of a slant mode I dominated tearing crack, where the crack tip flipping mechanism is made to engage by imposing a slight mode III, has recently been studied in Felter and Nielsen (2017) [Assisted crack tip flipping under Mode I thin sheet tearing, Europ. J. Mech. A/Solids, 64;2017:58–68]. In the previous study, the Gurson–Tvergaard–Needleman model was used in its original form to limit the model parameter space and facilitate a search for a set of parameter that allows flipping of the slant crack face to occur. It is well known that the adopted version of the Gurson model can predict the shear bands that travel in front of a mode I tearing crack and these are essential features to slant crack propagation — let alone the experimentally observed crack tip flipping phenomenon. In fact, assisted crack tip flipping was achieved with this numerical model set-up, but only within a very narrow parameter window. The present work adopts a phenomenological shear extended Gurson model that allows for a study of the J3 dependency in ductile fracture at engineering scale in an attempt to widen this parameter window. By running series of large-scale computations, where a ductile tearing crack propagates multiple plate thicknesses, the authors can demonstrate tearing modes for various combinations of strain hardening, initial void volume fraction, and shear parameter. The shear damage contribution is found to shift the engagement of the crack tip flipping mechanism toward lower values of the initial void volume fraction for all levels of strain hardening considered.

Histological analysis with white light microscopy of few micron-thick sections of tissue remains a gold standard for tissue biopsy. However, this approach requires a cumbersome multi-step preparation of the sample and relies heavily on the skills of a pathologist. In general, any tissue pathology (e.g. inflammation, fibrosis, cancer) affects the constituents of tissue and changes both optical properties of cells and extracellular matrix. We suggest using the images of transmittance, retardance and depolarization of unstained tissue samples, acquired with the Mueller microscopy, as an input data for automated diagnostic image segmentation. The potential of combining polarimetric data with non-linear microscopy data, such as Second Harmonic Generation (SHG) and Two Photon Emission Fluorescence (TPEF), is also explored for the diagnostic of tissue specific components (e. g. collagen, elastin). The unstained histological cuts of full thickness skin equivalents and cervical tissue of mouse were used for multi-modal microscopy imaging. First, the statistical approach was tested for a segmentation of the images of artificial skin using the images of retardance, depolarization, and transmitted unpolarized intensity. The algorithm of density-based spatial clustering of applications with noise (DBSCAN) clearly discriminates the regions of dermal and epidermal layers of skin cuts. The same approach was tested on the extended set of data including polarimetric, SHG and TPEF images of the histological cuts of mouse uterine cervix. The typical circumferential arrangement of collagen fibers around the cervical os as well as elastin lining of the internal cavities was observed on the segmented images. Merging polarimetric data with SHG data makes clear the appearance of fine structures in the cervical os region. The work is ongoing on testing different data post-processing algorithms for increasing both contrast and accuracy of tissue image segmentation, thus, paving the way for the digital histology for tissue diagnostic.

Significance : Definitive diagnostics of many diseases is based on the histological analysis of thin tissue cuts with optical white light microscopy. Extra information on tissue structural properties obtained with polarized light would help the pathologist to improve the accuracy of his diagnosis. Aim: We report on using Mueller matrix microscopy data, logarithmic decomposition, and polarized Monte Carlo (MC) modeling for qualitative and quantitative analysis of thin tissue cuts to extract the information on tissue microstructure that is not available with a conventional white light microscopy. Approach: Unstained cuts of human skin equivalents were measured with a custom-built liquid-crystal-based Mueller microscope in transmission configuration. To interpret experimental data, we performed the simulations with a polarized MC algorithm for scattering anisotropic media. Several optical models of tissue (spherical scatterers within birefringent host medium, and combination of spherical and cylindrical scatterers within either isotropic or birefringent host medium) were tested. Results: A set of rotation invariants for the logarithmic decomposition of a Mueller matrix was derived to rule out the impact of sample orientation. These invariants were calculated for both simulated and measured Mueller matrices of the dermal layer of skin equivalents. We demonstrated that only the simulations with a model combining both spherical and cylindrical scatterers within birefringent host medium reproduced the experimental trends in optical properties of the dermal layer (linear retardance, linear dichroism, and anisotropic linear depolarization) with layer thickness. Conclusions: Our studies prove that Mueller polarimetry provides relevant information not only on a size of dominant scatterers (e.g., cell nuclei versus subwavelength organelles) but also on its shape (e.g., cells versus collagen fibers). The latter is directly related to the state of extracellular collagen matrix, which is often affected by early pathology. Hence, using polarimetric data can help to increase the accuracy of diagnosis.

Crack tip flipping, where the fracture surface alternates from side to side in roughly 45° shear bands, seems to be an overlooked propagation mode in Mode I thin sheet tearing. In fact, observations of crack tip flipping is rarely found in the literature. Unlike the already established modes such as slanting, cup-cone (rooftop), or cup-cup (bathtub) the flipping crack never settles in a steady-state as the near tip stress/strain field continuously change when the flip successively initiates and develops shear-lips. A recent experimental investigation has revealed new insight by exploiting 3D X-ray tomography scanning of a developing crack tip flip. But, it remains to be understood what makes the crack flip systematically, what sets the flipping frequency, and under which material conditions this mode occurs. The present study aims at investigating the idea that a slight out-of-plane action (Mode III type loading) on the tip of a slant Mode I crack can provoke it to flip to the opposite side. Both experiments and micro-mechanics based modeling support this hypothesis.

Mueller transmission microscopy has been used for both theoretical and experimental studies of anisotropic scattering biological tissue. In our prior study, the linear dependence of retardance and quadratic dependence of depolarization on thickness was demonstrated for a dermal layer of skin model. During the primary analysis of polarimetric images of histological cuts both epidermal and dermal layers were delineated manually in order to calculate the spatially averaged values of retardance and depolarization parameters. Consequently, these average values contained the contribution of outliers (noise, not correctly identified pixels, etc.) which produces large standard deviation and biased mean values of the parameters mentioned above. For preventing the errors, the normalized maps of optical properties were calculated pixel-wise taking into account local optical density (e. i. logarithm of M11 element of Mueller matrix at each image pixel) to compensate varying tissue thickness across the cut area. Furthermore, the DBSCAN (Density-based spatial clustering of applications with noise) algorithm was applied for segmentation of microscopic images using the normalized values of retardance, depolarization, and intensity. From the results of image segmentation, we could discriminate the regions of dermal and epidermal layers in Muller microscopic images of skin cuts more accurately and obtain more reliable values of tissue’s optical properties.

The combined approach including experiments (Mueller polarimetry) and theory (differential Mueller matrix formalism) for the studies of anisotropic scattering media was tested on the model system of human skin. Custom-build Mueller polarimetric microscope was used for the studies of histological cuts of full-thickness skin equivalents generated from epidermal keratinocytes forming a multilayered epidermis on top of collagen I hydrogel with dermal fibroblasts. The sets of fixed unstained tissue cuts of different thicknesses (5μm - 30μm) were measured in transmission configuration. The values of polarimetric (dichroism and retardance) and depolarization parameters were calculated by applying pixel-wise the logarithmic decomposition of Mueller matrices. The parabolic dependence of depolarization parameters and linear dependence of polarimetric parameters on thickness, as predicted by theory, was confirmed by measurements. It proves that phenomenological modeling of complex anisotropic scattering medium (e.g., biological tissue) may effectively disentangle the polarimetric and depolarizing properties of the system understudies and may be used for analysis and diagnostics of tissue.

Because of a rising use of in vitro models as an alternative to animal models, the precise assessment of tissue-specific parameters of such in vitro test systems has become a critical part of ensuring predictive results. Impedance spectroscopy as a noninvasive method serves as a reliable and efficient tool for quality control because it only minimally interferes with the system during investigation. In this study, we present a refined impedance measurement system using nanostructured titanium nitride (TiN) electrodes. This advanced material was used to investigate tissue maturation and changes in the barrier integrity of an intestinal Transwell-based in vitro model. The reduction of noise facilitated a more detailed data extraction and biological interpretation. Compared to standard stainless steel electrodes, at a typical measurement frequency of 12.5 Hz, the maximum electrode impact on the signal could be reduced from over 75% to less than 5%. This allowed the accurate determination of transepithelial electrical resistance values from Caco-2 in vitro tissue models without a further mathematical analysis based on a computer simulation. The novel design of a 3D-printed measurement attachment equipped with nanostructured TiN electrodes was used to continuously monitor the barrier integrity of the Caco-2 cells during a permeability assay. Moreover, because of low process temperatures, the TiN coatings for enhanced impedance measurement sensitivity could also be deposited onto several other materials, e.g., commercially available cell culture equipment such as standard disposable multiwell plate dishes. In conclusion, we developed a novel method to improve the electrode properties for impedance spectroscopy, which can be easily implemented into standardized end-point measurement to qualify a variety of in vitro test systems.

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.

Mueller microscopy studies of fixed unstained histological cuts of human skin models were combined with an analysis of experimental data within the framework of differential Mueller matrix (MM) formalism. A custom-built Mueller polarimetric microscope was used in transmission configuration for the optical measurements of skin tissue model adjacent cuts of various nominal thicknesses (5 to 30 mm). The maps of both depolarization and polarization parameters were calculated from the corresponding microscopic MM images by applying a logarithmic Mueller matrix decomposition (LMMD) pixelwise. The parameters derived from LMMD of measured tissue cuts and the intensity of transmitted light were used for an automated segmentation of microscopy images to delineate dermal and epidermal layers. The quadratic dependence of depolarization parameters and linear dependence of polarization parameters on thickness, as predicted by the theory, was confirmed in our measurements. These findings pave the way toward digital histology with polarized light by presenting the combination of optimal optical markers, which allows mitigating the impact of tissue cut thickness fluctuations and increases the contrast of polarimetric images for tissue diagnostics.