Differential Interference Contrast Images Research Articles

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Single-exposure quantitative differential interference contrast microscopy using bandlimited image and its Fourier transform constraints

Phase microscopy that records the bandlimited image and its Fourier image simultaneously (BIFT) is a phase retrieval method with unique and rapid convergence. In this paper, we present a single-exposure quantitative differential interference contrast (DIC) microscopy based on BIFT method. The contrasts of the recorded DIC image and its Fourier image, analyzed by simulation and experiment, can be largely improved by the initial phase difference between two sheared lights (bias), however their trends with biases are opposite. By adding the optimized bias with the compromise of the contrasts in image and Fourier space, the phase sensitivity can be improved than BIFT method only. We have experimentally demonstrated that a sample of 25 nm height can be successfully recovered from a single exposure. The presented single-exposure quantitative DIC microscopy provides a promising technique for real-time phase imaging.

MTCSNet: One-stage learning and two-point labeling are sufficient for cell segmentation.

Deep convolution neural networks have been widely used in medical image analysis, such as lesion identification in whole-slide images, cancer detection, and cell segmentation, etc. However, it is often inevitable that researchers try their best to refine annotations so as to enhance the model performance, especially for cell segmentation task. Weakly supervised learning can greatly reduce the workload of annotations, while there is still a huge performance gap between the weakly and fully supervised learning approaches. In this work, we propose a weakly-supervised cell segmentation method, namely Multi-Task Cell Segmentation Network (MTCSNet), for multi-modal medical images, including pathological, brightfield, fluorescent, phase-contrast and differential interference contrast images. MTCSNet is learnt in a single-stage training manner, where only two annotated points for each cell provide supervision information, and the first one is the centroid, the second one is its boundary. Additionally, five auxiliary tasks are elaborately designed to train the network, including two pixel-level classifications, a pixel-level regression, a local temperature scaling and an instance-level distance regression task, which is proposed to regress the distances between the cell centroid and its boundaries in eight orientations. The experimental results indicate that our method outperforms all state-of-the-art weakly-supervised cell segmentation approaches on public multi-modal medical image datasets. The promising performance also shows that a single-stage learning with two-point labeling approach are sufficient for cell segmentation, instead of fine contour delineation. The codes are available at: https://github.com/binging512/MTCSNet.

A spatial carrier dynamic quantitative differential phase imaging method

Differential interference contrast (DIC) microscopy is an important method for quantitative phase imaging (QPI) due to its high compatibility regarding to partial coherent light illumination and excellent optical sectioning ability. However, limited by the on-axis interferometric configuration of Nomarski DIC microscopic imaging system, it is difficult to achieve single-shot phase retrieval. Here, we propose a simple and efficient method for dynamic quantitative differential phase imaging (QDPI) by introducing a spatial carrier device into DIC microscope. A Wollaston prism (WP) is placed on the imaging plane of DIC microscope and then projected by a 4f imaging system, so two orthogonally polarized imaging beams with a certain shearing amount will interfere on the conjugate plane of the 4f imaging system and form a spatial carrier DIC image, thus the QDPI can be conveniently realized. Importantly, the system not only possesses high temporal phase stability provided by the common-path geometries and low spatial phase noise due to partially coherent light illumination, but also the advantages of simple optical implementation, no additional phase distortion, high light energy utilization efficiency, and high signal-to-noise ratio. The experimental result demonstrates that using the proposed method, the phase measurement sensitivity reaches 0.003 rad, the corresponding applicability is verified by a polystyrene microsphere crown and biological cells.

Direct Observation of In-Focus Plasmonic Cargos via Breaking Angular Degeneracy in Differential Interference Contrast Microscopy.

Breaking the angular degeneracy arising from the 2-fold optical symmetry of plasmonic anisotropic nanoprobes is critical in biological studies. In this study, we propose differential interference contrast (DIC) microscopy-based focused orientation and position imaging (dFOPI) to break the angular degeneracy of single gold nanorods (AuNRs). Single in-focus AuNRs (39 nm × 123 nm) within a spherical mesoporous silica shell were characterized with high throughput and produced distinct doughnut-shaped DIC image patterns featuring two lobes in the peripheral region, attributed to the scattering contribution of the AuNRs with large scattering cross sections. Interestingly, rotation of the lobes was observed in the focal plane for a large AuNR (>100 nm) tilted by more than ∼20° from the horizontal plane as the rotational stage was moved by 10° in a rotational study. From the rotation-dependent characteristic patterns, we directly visualized counterclockwise/clockwise rotations without the angular degeneracy at the localized surface plasmon resonance wavelength. Therefore, our dFOPI method can be applied for in vivo studies of important biological systems. To validate this claim, we tracked the three-dimensional rotational behavior of transferrin-modified in-focus AuNRs during clathrin-mediated endocytosis in real time without sacrificing the temporal and spatial resolution. In the invagination and scission stage, one or two directed twist motions of the AuNR cargos detached the AuNR-containing vesicles from the cell membrane. Furthermore, the dFOPI method directly visualized and revealed the right-handed twisting action along the dynamin helix in dynamin-catalyzed fission in live cells.

Self-Association of Von Willebrand Factor (VWF) into Prothrombotic Fibers Is Most Potently Enhanced By the Highly Atherogenic Small, Dense Fraction of Low-Density Lipoprotein (sd-LDL)

We recently demonstrated that plasma lipoproteins play a major role in regulating the function of the hemostatic system by either inhibiting or promoting the self-association of von Willebrand factor (VWF) into large fibers and bundles that are hyperadhesive for platelets. Such fibers can form on the surface of activated endothelium such as during thrombotic microangiopathies. We showed that high-density lipoprotein (HDL) attenuates VWF self-association and reduces the formation of VWF-platelet thrombi, both in vitro and in mice (PMID:26552698). By contrast, low-density lipoprotein (LDL) enhances VWF self-association and promotes the formation of VWF-platelet thrombi ( Blood, in press). Consequently, wild-type mice with elevated plasma LDL display intense and prolonged VWF-platelet thrombosis in stimulated mesenteric microvessels, exceeding even the thrombosis observed in ADAMTS13-deficient mice with normal LDL levels. All the lipoproteins of low density carry apolipoprotein (apo) B100 and comprise fractions of very low density (VLDL), intermediate density (IDL), and low density (LDL). The LDL fraction comprises two subfractions: small-dense (sd) LDL and large-buoyant (lb) LDL. Epidemiologic studies suggest that sd-LDL is the most atherogenic subfraction (PMID:33586462). Another atherogenic lipoprotein also carries apoB-100, Lp(a), in which the apoB-100 is complexed with apo(a). Here, we sought to determine the effect of LDL subfraction(s) on VWF self-association. We isolated LDL, sd-LDL and lb-LDL from healthy donor plasma by sequential KBr density centrifugation. Lp(a) was purified from patients with high circulating Lp(a) (&amp;gt;50 mg/dL) as described by Mueller et al. (PMID:35650291). We first tested these lipoproteins in microfluidic devices adapted from the design of Herbig and Diamond (PMID:26178390). In these devices, high shear stress and flow acceleration are generated around a 30-um-wide pillar within a 60-um-wide flow channel and induce the attachment and self-association of VWF into fibers on and around the pillar. We quantified VWF fiber accumulation from differential interference contrast (DIC) images. Of all the lipoprotein fractions, sd-LDL was the most potent in accelerating VWF strand accumulation (see figure). The effect of lipoproteins in this device correlated with their impact on microvascular thrombosis in vivo. We infused apoB100-containing lipoproteins into wild-type mice (3-4 weeks old) before inducing VWF-platelet thrombi deposition in the mesenteric microvasculature with ionophore. All the apoB100 lipoproteins enhanced microvascular thrombosis [we did not test Lp(a), which had no effect in the microfluidic channel], but sd-LDL was by far the most potent; the treated mice had twice the number of large thrombi (&amp;gt;30-um diameter) as the mice treated with an equivalent quantity of lb-LDL. The effect also persisted for much longer before returning to baseline with the sd-LDL. We have explored possible reasons why sd-LDL is so much more potent. Compared to the other apoB-containing lipoproteins, sd-LDL is smaller and denser, with a greater surface curvature. Whether this fact changes accessibility to sites on apoB100 is not known. We also compared the lipidomes and proteomes of sd-LDL and lb-LDL for clues to the difference. Compared to lb-LDL, sd-LDL contained a much higher content of cholesterol and cholesterol esters per particle. The proteomes of the two particles were also different, with several proteins being more abundant in lb-LDL, in particular apoC-III, apoE, apoA-IV, and apoC-II. Only apoF was significantly more abundant in sd-LDL. We are currently testing to determine if any of these difference accounts for the differences between the two LDL subfractions in promoting VWF self-association. In summary, we show that the lipoprotein associated with the greatest atherosclerotic burden and most cardiovascular disease is also the one with the most potent effect in promoting a prothrombotic state by enhancing VWF self-association. These findings have important implications for the development of the atherosclerotic lesion, the formation of the thrombus after the lesion ruptures, and for the microvascular dysfunction often observed after revascularization.

A-144 Three-wavelength Polarization Imaging Method for the Analysis of Individual Human Blood Cells With Dye-Enhanced Dispersion of Dilution Buffer

Abstract Background Performing basic hematology analysis in a near-patient setting enables more rapid clinical decision making in the process of diagnosis. Traditional blood analyzers in central labs use a complex combination of sensing principles and chemical modification of the sample to derive the necessary parameters for a complete blood count (CBC). To reduce cost and maintenance, varying approaches have been employed to determine a CBC with a single-step measurement technique. One common option is the use of optical microscopy to deliver highly reliable results, examples are fluorescence microscopy, digital holographic microscopy, or spatial light interference microscopy. However, most of these technologies lack a proper determination of cellular-level RBC parameters. Therefore, a non-interferometric, quantitative-phase measurement technique was developed to derive relevant CBC parameters from a single measurement, comparable to a standard lab analyzer. Methods The optical setup employed by this method enables the parallel acquisition of brightfield and differential interference contrast (DIC) images by polarization-sensitive cameras. By using three cameras with individual optical filters, quantitative phase and absorption information can be calculated, which enables the determination of mean cellular volume (MCV), mean cellular hemoglobin (MCH), and mean cellular hemoglobin concentration (MCHC) for red blood cells (RBCs). The whole-blood sample is diluted in a buffer containing a non-cell-penetrating dye to enhance optical refraction contrast and therefore precision in the dependent calculated quantities. The sample is then introduced to a glass flow cell, and the cells are allowed to sediment to the bottom for imaging. Machine learning is applied to the derived images to classify unstained native white blood cells (WBCs) to yield a 5-part differential, which distinguishes between neutrophil, monocyte, lymphocyte, eosinophil and basophil. The algorithm is trained by imaging FACS-validated, purified WBC subgroups. All blood samples are measured on an ADVIA® 2120 Hematology System for control. Results A one-step, optical CBC hematology system based on quantitative phase and absorption image analysis was developed. The correlations for MCV and MCHC compared to a standard lab CBC analyzer were 0.77 and 0.84. The accuracy for the 5-part differential WBC classification was approximately 0.8. To enable a visual inspection of the classification results, a pseudo-colored image of each cell was generated and uploaded in a review tool. Conclusion With a more-refined, less-expensive optical setup, a high-quality CBC analyzer suitable for use at the point of care can be derived with the presented method. A possible simplification could be the use of low-cost but high-resolution Fourier ptychography microscopy (FPM) technology. Imaging of blood samples on a single-cell level offers the potential for the analysis of even more morphological parameters, for example, disease-related changes in the cell membrane. Also, the adhesion behavior of the WBCs in the glass flow cell could indicate cell activation status and therefore give information about infection processes. *Trademark disclaimer: “ADVIA, and all associated marks are trademarks of Siemens Healthcare Diagnostics, Inc. or its affiliates. All other trademarks and brands are the property of their respective owners.”

Cellsketch: Simplified Cell Representation for Label-free Cell and Nuclei Segmentation.

This paper presents a novel technique for cell segmentation, named "Cellsketch," which generates an RGB mask containing simplified representations of cells (including nuclei, whole-cell, and cell boundaries) from microscopic images, and applies the watershed algorithm to produce segmentation masks of cells and nuclei. The RGB mask is generated using a generator model trained with a combination of L1 loss and adversarial loss. The method achieved accurate cell and nuclei segmentation from differential interference contrast (DIC) images using only automatically annotated training data and shows potential for a generalizable algorithm for cell segmentation. The code is freely available at: https://github.com/iranovianti/cellsketchClinical Relevance- This method simultaneously detects individual cells and nuclei from DIC images.

Focus on lasers, imaging, microscopy, and nanoscience

Focus on lasers, imaging, microscopy, and nanoscience

Off-axis differential interference contrast (DIC) microscopy enabled by polarization gratings pair and synthetic aperture technology for integrational and sectional quantitative phase imaging

Differential interference contrast (DIC) microscopy is an important member of quantitative phase imaging (QPI) family owing to its simple optical implementation and high compatibility regarding to different coherence degree of illumination field. Such compactivity means that the DIC-based phase imaging can be conveniently converted between 2D-integrational and 3D-sectional modes by switching the spatial coherence degree of illumination field between low degree and high. However, there is a potential trade-off compromise in the existed experimental implementations for DIC imaging regarding to the optical simplicity, space-bandwidth utilization and temporal resolution. In this manuscript, we proposed a polarization gratings pair (PGs) based off-axis DIC system to realize integrational and sectional QPI. Building on a bright field microscope and getting rid of the additional 4f lens system, the proposed PGs-DIC system can realize single-shot QPI with an ultra-compact optical configuration. In order to improve the imaging resolution and optical sectional ability, a fast synthetic aperture technology is introduced, in which multiple interferograms with different line-filtered illumination are captured. Both high resolution 2D-integrational and 3D-sectional phase imaging results indicate that the proposed PGs-DIC system is a good candidate for QPI. Specially, a theoretical model is proposed to quantitatively estimate z-direction resolution for z-scanning 3D phase imaging technology and future application in the PGs-DIC system.

A cytoskeletal vortex drives phage nucleus rotation during jumbo phage replication in E.coli.

Nucleus-forming jumbo phages establish an intricate subcellular organization, enclosing phage genomes within a proteinaceous shell called the phage nucleus. During infection in Pseudomonas, some jumbo phages assemble a bipolar spindle of tubulin-like PhuZ filaments that positions the phage nucleus at midcell and drives its intracellular rotation. This facilitates the distribution of capsids on its surface for genome packaging. Here we show that the Escherichia coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of a mutant PhuZ protein strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes for cytoplasmic streaming and nucleus alignment, this work identifies a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

Two-dimensional measurement of resonance in MEMS resonators using stroboscopic differential interference contrast microscopy.

We report the two-dimensional (2D) measurement of resonance in MEMS resonators using stroboscopic differential interference contrast (DIC) microscopy, for the investigation of the linear and nonlinear oscillations of MEMS resonators. The DIC microscopy measures the interference of two sheared illumination light beams reflected from the sample surface to determine the differential surface deflection. By modulating the illumination light at the resonance frequency, the DIC image of the MEMS resonator periodically change its brightness and contrast with the sweeping illumination phase, which have been used to derive the oscillation amplitude and the resonance mode shape of the MEMS resonator. Comparing with conventional interference microscopy, the DIC microscopy can observe the surface deflection larger than the wavelength of the illumination light, enabling the measurement of nonlinear oscillations with a large oscillation amplitude. We demonstrate that the stroboscopic DIC microscopy can measure the 2D mechanical resonance with a high vertical resolution at the nanometer(nm)-scale, and a large measurement range of ∼1µm, which is very promising for the investigation of linear and nonlinear oscillations of MEMS resonators.

Slip slidin' away: Bristle-driven gliding by Tetradesmus deserticola (Chlorophyta) in microfluidic chambers.

Microalgae within the Scenedesmaceae are often distinguished by spines, bristles, and other wall characteristics. We examined the dynamic production and chemical nature of bristles extruded from the poles of Tetradesmus deserticola previously isolated from microbiotic crust. Rapidly growing cells in a liquid growth medium were established in polydimethylsiloxane microfluidic chambers specially designed to maintain aerobic conditions over time within a chamber 6-12 μm deep. This geometry enabled in-focus imaging of single cells over long periods. Differential interference contrast (DIC) imaging revealed that after multiple fission of mother cells, the newly released, lemon-shaped daughter cells began extruding bristles from each pole. In some instances, the bristles became stuck to either the glass floor or polydimethylsiloxane (PDMS) walls of the chamber, and the force by which the new bristle was extruded was sufficient to propel the cells across the field of view at ~1.2 μm · h-1 . Confocal fluorescence and DIC imaging of cells stained with pontamine fast scarlet and calcofluor, and treated with proteinase K, suggested that bristles are proteinaceous and may also host carbohydrate modifications. The polar bristles extruded by this desert-derived T. deserticola may simply be relics of bristles produced by an aquatic ancestor for flotation or predator deterrence. But, their tendency to attach to glass (silicate) and/or PDMS surfaces suggests a potential role in tethering cells in place or binding soil particles. T. deserticola is closely related to T. obliquus, which is of interest for biofuels development; extruded bristles in T. deserticola may offer tethers for industrial use of these stress-tolerant algae.

Full-field differential interference contrast imaging with radial-shearing Greek-ladder sieves

The traditional differential interference contrast (DIC) technique is an effective method of quantitative phase imaging, but it has the specific property of orientation and direction selectivity, which decreases the contrast of images in some degree. Here an approach of Radial-shearing DIC (RsDIC) is proposed to realize phase imaging of omnidirectional selectivity as well as high sensitivity, in which the key is an amplitude-only ancient Greek-ladder sieve. Two setups are proposed. One contains only one sieve, in which the interference occurs in the overlap region near to the two adjacent image planes. The other contains two confocal sieves, in which the interference occurs in the coincident image plane. The effectiveness of our proposed method is demonstrated by preliminary results obtained from the software GLAD simulation and experiment performed in visible light. RsDIC allows for full-field DIC imaging as well as for DIC scanning transmission x-ray microscopy, which can also provide a nanometer resolution nondestructive imaging tool in x-ray region for ultra-large-scale integrated circuits, living cells, etc.

Algorithm based on the 1-norm character of interferograms for quantitative differential interference imaging

Differential interference contrast (DIC) microscopy is widely used as a high-contrast imaging method for observing transparent biological samples. However, the absence of fringes in DIC images results in phase gradients that cannot be effectively extracted by most phase-shift algorithms, limiting its application in quantitative detection. Therefore, we propose an algorithm based on the 1-norm character of interferograms that can accurately extract phase shifts from a series of interferograms with less than 1/2 fringes. We improved this algorithm by combining the calculated phase shift with the least-squares fitting method to further extract the phase and applied it to quantitative DIC imaging. In contrast to the conventional algorithm, this algorithm can extract the phase gradient in DIC images with unknown phase shifts, thereby eliminating the computational error in the conventional algorithm introduced by imprecise phase shifts. Simulations and experiments demonstrated that the proposed algorithm can perform accurate phase gradient extraction.

Automating Pitted Red Blood Cell Counts Using Deep Neural Network Analysis: A New Method for Measuring Splenic Function in Sickle Cell Anaemia.

The spleen plays an important role in the body’s defence against bacterial infections. Measuring splenic function is of interest in multiple conditions, including sickle cell anaemia (SCA), where spleen injury occurs early in life. Unfortunately, there is no direct and simple way of measuring splenic function, and it is rarely assessed in clinical or research settings. Manual counts of pitted red blood cells (RBCs) observed with differential interference contrast (DIC) microscopy is a well-validated surrogate biomarker of splenic function. The method, however, is both user-dependent and laborious. In this study, we propose a new automated workflow for counting pitted RBCs using deep neural network analysis. Secondly, we assess the durability of fixed RBCs for pitted RBC counts over time. We included samples from 48 children with SCA and 10 healthy controls. Cells were fixed in paraformaldehyde and examined using an oil-immersion objective, and microscopy images were recorded with a DIC setup. Manual pitted RBC counts were performed by examining a minimum of 500 RBCs for pits, expressing the proportion of pitted RBCs as a percentage (%PIT). Automated pitted RBC counts were generated by first segmenting DIC images using a Zeiss Intellesis deep learning model, recognising and segmenting cells and pits from background. Subsequently, segmented images were analysed using a small ImageJ macro language script. Selected samples were stored for 24 months, and manual pitted RBC counts performed at various time points. When comparing manual and automated pitted RBC counts, we found the two methods to yield comparable results. Although variability between the measurements increased with higher %PIT, this did not change the diagnosis of asplenia. Furthermore, we found no significant changes in %PIT after storing samples for up to 24 months and under varying temperatures and light exposures. We have shown that automated pitted RBC counts, produced using deep neural network analysis, are comparable to manual counts, and that fixed samples can be stored for long periods of time without affecting the %PIT. Automating pitted RBC counts makes the method less time consuming and results comparable across laboratories.

Differential phase measurement based on synchronous phase shift determination.

Based on synchronous phase shift determination, we propose a differential phase measurement method for differential interference contrast (DIC) microscopy. An on-line phase shift measurement device is used to generate carrier interferograms and determine the phase shift of DIC images. Then the differential phase can be extracted with the least-squares phase-shifting algorithm. In addition to realizing on-line, dynamic, real-time, synchronous and high precision phase shift measurement, the proposed method also can reconstruct the phase of the specimen by using the phase-integral algorithm. The differential phase measurement method reveals obvious advantages in error compensation, anti-interference, and noise suppression. Both simulation analysis and experimental result demonstrate that using the proposed method, the accuracy of phase shift measurement is higher than 0.007 rad. Very accurate phase reconstructions were obtained with both polystyrene microspheres and human vascular endothelial.

Fully automated platelet differential interference contrast image analysis via deep learning

Platelets mediate arterial thrombosis, a leading cause of myocardial infarction and stroke. During injury, platelets adhere and spread over exposed subendothelial matrix substrates of the damaged blood vessel wall. The mechanisms which govern platelet activation and their interaction with a range of substrates are therefore regularly investigated using platelet spreading assays. These assays often use differential interference contrast (DIC) microscopy to assess platelet morphology and analysis performed using manual annotation. Here, a convolutional neural network (CNN) allowed fully automated analysis of platelet spreading assays captured by DIC microscopy. The CNN was trained using 120 generalised training images. Increasing the number of training images increases the mean average precision of the CNN. The CNN performance was compared to six manual annotators. Significant variation was observed between annotators, highlighting bias when manual analysis is performed. The CNN effectively analysed platelet morphology when platelets spread over a range of substrates (CRP-XL, vWF and fibrinogen), in the presence and absence of inhibitors (dasatinib, ibrutinib and PRT-060318) and agonist (thrombin), with results consistent in quantifying spread platelet area which is comparable to published literature. The application of a CNN enables, for the first time, automated analysis of platelet spreading assays captured by DIC microscopy.

Subchondral bone plate thickness is associated with micromechanical and microstructural changes in the bovine patella osteochondral junction with different levels of cartilage degeneration

The influence of joint degeneration on the biomechanical properties of calcified cartilage and subchondral bone plate at the osteochondral junction is relatively unknown. Common experimental difficulties include accessibility to and visualization of the osteochondral junction, application of mechanical testing at the appropriate length scale, and availability of tissue that provides a consistent range of degenerative changes. This study addresses these challenges.A well-established bovine patella model of early joint degeneration was employed, in which micromechanical testing of fully hydrated osteochondral sections was carried out in conjunction with high-resolution imaging using differential interference contrast (DIC) optical light microscopy. A total of forty-two bovine patellae with different grades of tissue health ranging from healthy to mild, moderate, and severe cartilage degeneration, were selected. From the distal–lateral region of each patella, two adjacent osteochondral sections were obtained for the mechanical testing and the DIC imaging, respectively. Mechanical testing was carried out using a robotic micro-force acquisition system, applying compression tests over an array (area: 200 μm × 1000 μm, step size: 50 μm) across the osteochondral junction to obtain a stiffness map. Morphometric analysis was performed for the DIC images of fully hydrated cryo-sections. The levels of cartilage degeneration, DIC images, and the stiffness maps were used to associate the mechanical properties onto the specific tissue regions of cartilage, calcified cartilage, and subchondral bone plate.The results showed that there were up to 20% and 24% decreases (p < 0.05) in the stiffness of calcified cartilage and subchondral bone plate, respectively, in the severely degenerated group compared to the healthy group. Furthermore, there were increases (p < 0.05) in the number of tidemarks, bone spicules at the cement line, and the mean thickness of the subchondral bone plate with increasing levels of degeneration.The decreasing stiffness in the subchondral bone plate coupled with the presence of bone spicules may be indicative of a subchondral remodeling process involving new bone formation. Moreover, the mean thickness of the subchondral bone plate was found to be the strongest indicator of mechanical and associated structural changes in the osteochondral joint tissues.

Shear direction switching phenomena in twisted nematic liquid crystal cells for novel differential interference contrast imaging systems

AbstractA lateral shear occurs between ordinary and extraordinary rays in general optical uniaxial crystals, and we can observe a double image through the crystal, indicating a typical birefringence phenomenon. Small lateral shears occur in typical liquid crystal (LC) cells under small voltage applications, where the molecular alignment direction is generally inclined. The small lateral shear is useful for differential interference contrast (DIC) imaging systems, although it must be negligible in a typical LC display panel. In particular, unique lateral shearing properties are exhibited by twisted nematic (TN) LC cells, which are popular and important LC display modes. The 45°‐oblique lateral shear occurs according to the direction of the surface molecular alignment, and the inclination direction (+45° or −45°) depends on the molecular twist direction. If it is possible to switch the twist direction in the TN cell, the lateral shear direction can be switched by 90°, and a full DIC imaging system can be attained because the normal DIC system can obtain one‐directional derivative information along the shear direction. In this work, twist direction switching phenomena in TN cells are investigated by applying a magnetic field in the direction parallel to the cell surface.