Influence of hematoxylin and eosin staining on linear birefringence measurement of fibrous tissue structures in polarization microscopy.

Significance: For microscopic polarization imaging of tissue slices, two types of samples are often prepared: one unstained tissue section for polarization imaging to avoid possible influence from staining dyes quantitatively and one hematoxylin-eosin (H&E) stained adjacent tissue section for histological diagnosis and structural feature identification. However, this sample preparation strategy requires high-quality adjacent tissue sections, and labeling the structural features on unstained tissue sections is impossible. With the fast development of data driven-based polarimetric analysis, which requires a large amount of pixel labeled images, a possible method is to directly use H&E stained slices, which are standard samples archived in clinical hospitals for polarization measurement. Aim: We aim to study the influence of hematoxylin and eosin staining on the linear birefringence measurement of fibrous tissue structures. Approach: We examine the linear birefringence properties of four pieces of adjacent bone tissue slices with abundant collagen fibers that are unstained, H&E stained, hematoxylin (H) stained, and eosin (E) stained. After obtaining the spatial maps of linear retardance values for the four tissue samples, we carry out a comparative study using a frequency distribution histogram and similarity analysis based on the Bhattacharyya coefficient to investigate how H&E staining affects the linear birefringence measurement of bone tissues. Results: Linear retardance increased after H&E, H, or E staining (41.7%, 40.8%, and 72.5% increase, respectively). However, there is no significant change in the imaging contrast of linear retardance in bone tissues. Conclusions: The linear retardance values induced by birefringent collagen fibers can be enhanced after H&E, H, or E staining. However, the structural imaging contrasts based on linear retardance did not change significantly or the staining did not generate linear birefringence on the sample area without collagen. Therefore, it can be acceptable to prepare H&E stained slices for clinical applications of polarimetry based on such a mapping relationship.

Determination of Light-Induced Deformation Characteristics of the Director Field of Nematic Liquid Crystal by Combining the Methods of Aberrational Self-Action of Light Beam and Polarization Microscopy.

Light-induced director field deformation of a nematic liquid crystal in the field of an obliquely incident laser beam is experimentally studied with aberrational self-action and polarization microscopy. Each of the methods has features associated with the geometry of the light interaction with the director. The combination of methods significantly expands the possibilities of reconstructing the light-induced nonlinear phase shift profile and the director field deformation.

Dark-field light scattering microscope with focus stabilization.

We present detailed design and operation instructions for a single-objective inverted microscope. Our design is suitable for two dark-field modes of operation: 1- total internal reflection scattering, and 2- cross-polarization backscattering. The user can switch between the two modes by exchanging one mode-steering element, which is also adapted to the Thorlabs cage system. To establish a stable background speckle for differential microscopy the imaging plane is stabilized with active feedback. We validate the stabilization efficacy by performing long-term scattering measurement on single nanoparticles. This setup can be extended for simultaneous scattering, fluorescence, and confocal imaging modes.

High-Performance Polarization Microscopy Reveals Structural Remodeling in Rat Calcaneal Tendons Cultivated In Vitro.

Collagenous tissues exhibit anisotropic optical properties such as birefringence and linear dichroism (LD) as a result of their structurally oriented supraorganization from the nanometer level to the collagen bundle scale. Changes in macromolecular order and in aggregational states can be evaluated in tendon collagen bundles using polarization microscopy. Because there are no reports on the status of the macromolecular organization in tendon explants, the objective of this work was to evaluate the birefringence and LD characteristics of collagen bundles in rat calcaneal tendons cultivated in vitro on substrates that differ in their mechanical stiffness (plastic vs. glass) while accompanying the expected occurrence of cell migration from these structures. Tendon explants from adult male Wistar rats were cultivated for 8 and 12 days on borosilicate glass coverslips (n = 3) and on nonpyrogenic polystyrene plastic dishes (n = 4) and were compared with tendons not cultivated in vitro (n = 3). Birefringence was investigated in unstained tendon sections using high-performance polarization microscopy and image analysis. LD was studied under polarized light in tendon sections stained with the dichroic dyes Ponceau SS and toluidine blue at pH 4.0 to evaluate the orientation of proteins and acid glycosaminoglycans (GAG) macromolecules, respectively. Structural remodeling characterized by the reduction in the macromolecular orientation, aggregation and alignment of collagen bundles, based on decreased average gray values concerned with birefringence intensity, LD and morphological changes, was detected especially in the tendon explants cultivated on the plastic substrate. These changes may have facilitated cell migration from the lateral regions of the explants to the substrates, an event that was observed earlier and more intensely upon tissue cultivation on the plastic substrate. The axial alignment of the migrating cells relative to the explant, which occurred with increased cultivation times, may be due to the mechanosensitive nature of the tenocytes. Collagen fibers possibly played a role as a signal source to cells, a hypothesis that requires further investigation, including studies on the dynamics of cell membrane receptors and cytoskeletal organization, and collagen shearing electrical properties.

Polarization clustering of biological structures with Mueller matrix parameters.

Mueller matrix imaging polarimetry (MMIP) is a promising technique for the characterization of biological tissues, including the classification of microstructures in pathological diagnosis. To expand the parameter space of Mueller matrix parameters, we propose new vector parameters (VPs) according to the Mueller matrix polar decomposition method. We measure invasive bladder cancer (IBC) with extensive necrosis and high-grade ductal carcinoma in situ (DCIS) with MMIP, and the regions of cancer cells and fibrotic stroma are classified with the VPs. Then the proposed and existing VPs are mapped on the Poincaré sphere with 3D visualization, and an indicator of spatial feature is defined based on the minimum enclosing sphere to evaluate the classification capability of the VPs. For both IBC and DCIS, the results show that the proposed VPs exhibit evident contrast between the regions of cancer cells and fibrotic stroma. This study broadens the fundamental Mueller matrix parameters and helps to improve the characterization ability of the MMIP technique.

Periphyton (microbiome) of filamentous algae studied by polarization microscopy, aided also by some contrasting optical methods.

Polarization microscopy, possibly together with some contrast techniques (dark field and color phase contrast), was used to study the periphyton (microbiome) growing on filamentous green algae. The material containing filamentous algae with periphyton on the surface was collected in the villages of Sýkorice and Zbecno (Krivoklátsko Protected Landscape Area). The objects were studied in a LOMO MIN-8 St. Petersburg polarizing microscope and a Carl Zeiss Jena NfpK laboratory microscope equipped with an In Ph 160 basic body with variable dark field or color phase contrast and a Nikon D70 DSLR digital camera. Cells of filamentous algae of the genera Cladophora, Vaucheria, and Oedogonium were studied and the periphyton attached to them formed by cyanobacteria of the genera Chamaesiphon and Pleurocapsa and algae of the genera Characium, including diatoms of the genera Eunotia and Synedra. In all cases, the cell walls of the host algae showed a very strong birefringence. In contrast, the walls of cyanobacteria of the genera Chamaesiphon and Pleurocapsa were characterized by a much weaker birefringence (Pleurocapsa somewhat thicker), and the diatom frustules of the genera Eunotia and Synedra were almost without a birefringence. Strongly birefringent granules were found in the cytoplasm of the green alga of the genus Characium, which forms periphyton on the filamentous green algae of the genus Vaucheria. The periphyton on the filamentous alga of the genus Oedogonium, formed by cyanobacteria of the genus Pleurocapsa and diatoms of the genera Eunotia and Synedra, deposited in a massive layer of mucus containing birefringent crystals, showed a particularly strong birefringence. At the end of the vegetation of filamentous algae, their parts and remnants of periphyton (diatom frustules and crystals) became part of the detritus at the bottom of the culture vessel. The use of polarization microscopy in the study of filamentous algae with periphyton on the surface allows us not only to determine the birefringence of the observed structures, but also to partially deduce their chemical composition, or regular arrangement of particles, so-called shape birefringence.

Study of the spatial scale stability of Mueller matrix parameters for textural characterization of biological tissues.

Mueller matrix imaging polarimetry (MMIP) is a promising technique for the textural characterization of biological tissue structures. To reveal the influence of imaging magnification on the robustness of Mueller matrix parameters (MMPs), the spatial scale stability of MMPs was studied. We established a new MMIP detector and derived the mathematical model of the spatial scale stability of MMPs. The biological tissues with well-defined structural components were imaged under different magnifications. Then, we compared and analyzed the textural features of the MMPs in the resulting images. The experimental results match the predictions of the mathematical model in these aspects: (a) magnification exhibits a strong nonlinear effect on the textural contrasts of MMPs images; (b) higher magnification does not necessarily lead to superior contrast for textural characterization; and (c) for different biological tissues, MMPs contrasts can be optimized differently, with some showing superior results. This study provides a reference for the experimental design and operation of the MMIP technique and is helpful for improving the characterization ability of MMPs.

Dynamic Instability of Individual Carbon Nanotube Growth Revealed by In Situ Homodyne Polarization Microscopy.

Understanding the kinetic selectivity of carbon nanotube growth at the scale of individual nanotubes is essential for the development of high chiral selectivity growth methods. Here we demonstrate that homodyne polarization microscopy can be used for high-throughput imaging of long individual carbon nanotubes under real growth conditions (at ambient pressure, on a substrate) and with subsecond time resolution. Our in situ observations on hundreds of individual nanotubes reveal that about half of them grow at a constant rate all along their lifetime while the other half exhibits stochastic changes in growth rates and/or switches between growth, pause, and shrinkage. Statistical analysis shows that the growth rate of a given nanotube essentially varies between two values, with a similar average ratio (∼1.7) regardless of whether the rate change is accompanied by a change in chirality. These switches indicate that the nanotube edge or the catalyst nanoparticle fluctuates between different configurations during growth.

Optical sensors of heterotrimeric G protein signaling.

Heterotrimeric G proteins are central mediators of cellular signal transduction. They receive, process, and transduce signals from G protein-coupled receptors to downstream effectors. Since their discovery, a number of optical sensors of G protein localisation and function have been developed and applied in living systems. In this minireview, we provide an overview of existing G protein-based sensors and the experimental approaches they utilise, with emphasis on live-cell imaging techniques. We outline recent advances, as well as identify current challenges and likely future directions in the field of G protein sensor development.

Single-Shot Quantitative Polarization Imaging of Complex Birefringent Structure Dynamics.

Polarization light microscopes are powerful tools for probing molecular order and orientation in birefringent materials. While a number of polarization microscopy techniques are available to access steady-state properties of birefringent samples, quantitative measurements of the molecular orientation dynamics on the millisecond time scale have remained a challenge. We propose polarized shearing interference microscopy (PSIM), a single-shot quantitative polarization imaging method, for extracting the retardance and orientation angle of the laser beam transmitting through optically anisotropic specimens with complex structures. The measurement accuracy and imaging performance of PSIM are validated by imaging a birefringent resolution target and a bovine tendon specimen. We demonstrate that PSIM can quantify the dynamics of a flowing lyotropic chromonic liquid crystal in a microfluidic channel at an imaging speed of 506 frames per second (only limited by the camera frame rate), with a field-of-view of up to 350 × 350 µm2 and a diffraction-limit spatial resolution of ~2 µm. We envision that PSIM will find a broad range of applications in quantitative material characterization under dynamical conditions.

Conformation of the nuclear pore in living cells is modulated by transport state.

While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, in vivo conformational changes of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.

Directionality of light absorption and emission in representative fluorescent proteins.

Fluorescent molecules are like antennas: The rate at which they absorb light depends on their orientation with respect to the incoming light wave, and the apparent intensity of their emission depends on their orientation with respect to the observer. However, the directions along which the most important fluorescent molecules in biology, fluorescent proteins (FPs), absorb and emit light are generally not known. Our optical and X-ray investigations of FP crystals have now allowed us to determine the molecular orientations of the excitation and emission transition dipole moments in the FPs mTurquoise2, eGFP, and mCherry, and the photoconvertible FP mEos4b. Our results will allow using FP directionality in studies of molecular and biological processes, but also in development of novel bioengineering and bioelectronics applications.

Advances of super-resolution fluorescence polarization microscopy and its applications in life sciences.

Fluorescence polarization microscopy (FPM) analyzes both intensity and orientation of fluorescence dipole, and reflects the structural specificity of target molecules. It has become an important tool for studying protein organization, orientational order, and structural changes in cells. However, suffering from optical diffraction limit, conventional FPM has low orientation resolution and observation accuracy, as the polarization information is averaged by multiple fluorescent molecules within a diffraction-limited volume. Recently, novel super-resolution FPMs have been developed to break the diffraction barrier. In this review, we will introduce the recent progress to achieve sub-diffraction determination of dipole orientation. Biological applications, based on polarization analysis of fluorescence dipole, are also summarized, with focus on chromophore-target molecule interaction and molecular organization.

Advantages of Fresnel biprism-based digital holographic microscopy in quantitative phase imaging.

SIGNIFICANCE: The hallmarks of digital holographic microscopy (DHM) compared with other quantitative phase imaging (QPI) methods are high speed, accuracy, spatial resolution, temporal stability, and polarization-sensitivity (PS) capability. The above features make DHM suitable for real-time quantitative PS phase imaging in a broad number of biological applications aimed at understanding cell growth and dynamic changes occurring during physiological processes and/or in response to pharmaceutical agents. AIM: The insertion of a Fresnel biprism (FB) in the image space of a light microscope potentially turns any commercial system into a DHM system enabling QPI with the five desired features in QPI simultaneously: high temporal sensitivity, high speed, high accuracy, high spatial resolution, and PS. To the best of our knowledge, this is the first FB-based DHM system providing these five features all together. APPROACH: The performance of the proposed system was calibrated with a benchmark phase object. The PS capability has been verified by imaging human U87 glioblastoma cells. RESULTS: The proposed FB-based DHM system provides accurate phase images with high spatial resolution. The temporal stability of our system is in the order of a few nanometers, enabling live-cell studies. Finally, the distinctive behavior of the cells at different polarization angles (e.g., PS capability) can be observed with our system. CONCLUSIONS: We have presented a method to turn any commercial light microscope with monochromatic illumination into a PS QPI system. The proposed system provides accurate quantitative PS phase images in a new, simple, compact, and cost-effective format, thanks to the low cost (a few hundred dollars) involved in implementing this simple architecture, enabling the use of this QPI technique accessible to most laboratories with standard light microscopes.

Advanced microscopy techniques for revealing molecular structure of starch granules.

Starch is a major source of our daily diet and it is important to understand the molecular structure that plays a significant role in its wide number of applications. In this review article, microscopic structures of starch granules from potato, corn, rice canna, tania, wheat, sweet potato, and cassava are revealed using advanced microscopic techniques. Optical microscopy depicts the size and shape, polarization microscopy shows the anisotropy properties of starch granules, scanning electron microscopy (SEM) displays surface topography, and confocal microscopy is used to observe the three-dimensional internal structure of starch granules. The crystallinity of starch granules is revealed by second harmonic generation (SHG) microscopy and atomic force microscopy (AFM) provides mechanical properties including strength, texture, and elasticity. These properties play an important role in understanding the stability of starch granules under various processing conditions like heating, enzyme degradation, and hydration and determining its applications in various industries such as food packaging and textile industries.

Deep Learning-Based Holographic Polarization Microscopy.

Polarized light microscopy provides high contrast to birefringent specimen and is widely used as a diagnostic tool in pathology. However, polarization microscopy systems typically operate by analyzing images collected from two or more light paths in different states of polarization, which lead to relatively complex optical designs, high system costs, or experienced technicians being required. Here, we present a deep learning-based holographic polarization microscope that is capable of obtaining quantitative birefringence retardance and orientation information of specimen from a phase-recovered hologram, while only requiring the addition of one polarizer/analyzer pair to an inline lensfree holographic imaging system. Using a deep neural network, the reconstructed holographic images from a single state of polarization can be transformed into images equivalent to those captured using a single-shot computational polarized light microscope (SCPLM). Our analysis shows that a trained deep neural network can extract the birefringence information using both the sample specific morphological features as well as the holographic amplitude and phase distribution. To demonstrate the efficacy of this method, we tested it by imaging various birefringent samples including, for example, monosodium urate and triamcinolone acetonide crystals. Our method achieves similar results to SCPLM both qualitatively and quantitatively, and due to its simpler optical design and significantly larger field-of-view this method has the potential to expand the access to polarization microscopy and its use for medical diagnosis in resource limited settings.

Collagen Organization Within the Cartilage of Trpv4-/- Mice Studied with Two-Photon Microscopy and Polarized Second Harmonic Generation.

The polymodal channel TRPV4 has been shown to regulate development and maintenance of cartilage. Here we investigate whether TRPV4 activity regulates the early deposition and structure of collagen matrix in the femoral head cartilage by comparing the 3D morphology and the sub-micrometer organization of the collagen matrix between wild type and Trpv4 -/- mice pups four to five days old. Two-photon microscopy can be used to conduct label-free imaging of cartilage, as collagen generates a second harmonic signal (second harmonic generation [SHG]) under pulsed infrared excitation. In one set of measurements, we use circularly polarized laser light to reconstruct the 3D morphology of the femoral head cartilage and to measure the tissue thickness. Second, by rotating the direction of the linearly polarized light and using polarized SHG detection, we investigate the sub-micrometer orientation of collagen fibers in the cartilage. At this developmental stage, we cannot detect statistically significant differences between the two mice strains, although a tendency toward a more random orientation of collagen fibers and a higher thickness of the whole cartilage seems to characterize the Trpv4 -/- mice. We discuss possible reasons for these observations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Origins of a pervasive, erroneous idea: The "green birefringence" of Congo red-stained amyloid.

Congo red was discovered to stain amyloid by accident in 1922, and Congo red-stained amyloid was shown to be birefringent on polarization microscopy in 1927. Colours, namely green and yellow, were reported under these conditions in 1945, although these are only two of various anomalous colours that may be seen, depending on the optical set-up. In 1953 there began a dogmatic insistence that in Congo red-stained amyloid between crossed polarizer and analyser green alone should be seen, and the finding of any other colour was a mistake. The idea that green, and only green, is essential for the diagnosis of amyloid has persisted almost universally, and virtually all mentions of Congo red-stained amyloid say that it just shows "green birefringence" or "apple-green birefringence." This idea is wrong and is contrary to everyday experience, because green is seldom seen on its own under these conditions of microscopy, and often, there is no green at all. How observers maintain this unscientific position is explained by a study of its historical origins. Most of the early literature was in German or French and was usually quoted in English at second hand, which meant that misquotations, misattributions and misunderstandings were common. Few workers reported their findings accurately, hardly any attempted to explain them, and until 2008, none gave a completely satisfactory account of the physical optics. The history of Congo red-stained amyloid is an instructive example of how an erroneous belief can become widely established even when it is contradicted by simple experience.

Synergic Interactions Between Hepatic Stellate Cells and Uveal Melanoma in Metastatic Growth.

Uveal melanoma (UM) is a malignant intraocular tumor that spreads to the liver in half of the cases. Since hepatic cells could play a role in the therapeutic resistance of metastatic UM, the purpose of our study was to investigate the pro-invasive role of hepatic stellate cells (HSteCs) in metastatic UM at the micro- and macro-metastatic stages. We first performed an immunostaining with the alpha-smooth muscle actin (αSMA) to localize activated HSteCs in UM liver macro-metastases from four patients. Their accumulation of collagen was assessed with Masson's Trichrome stain. Next, we inoculated metastatic UM cells alone or with human HSteCs in triple-immunodeficient mice, in order to determine if HSteCs are recruited as early as the micro-metastatic stage. The growth of metastatic foci was imaged in the liver by ex vivo fluorescence imaging. Histological analyses were performed with Masson's Trichrome and Picrosirius Red stains, and antibodies against Melan-A and αSMA. The collagen content was measured in xenografts by quantitative polarization microscopy. In patient hepatectomy samples, activated HSteCs and their pathological matrix were localized surrounding the malignant lesions. In the mouse xenograft model, the number of hepatic metastases was increased when human HSteCs were co-inoculated. Histological analyses revealed a significant recruitment of HSteCs near the micro/macrolesions, and an increase in fibrillar collagen production. Our results show that HSteCs can provide a permissive microenvironment and might increase the therapeutic resistance of metastatic UM.

Método químico simplificado de desmineralización del esmalte dental humano / Simplified chemical method of demineralization in human dental enamel

Introducción: Se requieren métodos experimentales abreviados para simular las lesiones de desmineralización temprana de forma controlada y reproducible. Objetivo: Realizar una evaluación in vitro de un método simple de desmineralización incipiente del esmalte. Métodos: Estudio experimental aleatorizado con doble diseño factorial de réplicas. Se seleccionaron 12 terceros molares de sujetos humanos saludables para su desmineralización en solución de ácido láctico racémico. Las muestras se distribuyeron aleatoriamente: Grupo 1 (G1) (n= 6) ácido láctico a pH 2,4 y Grupo 2 (G2) (n= 6) ácido láctico a pH 5,4. A continuación, cada grupo se subdividió (n = 2) para evaluar el efecto de las soluciones a tres tiempos de exposición (7, 15 y 30 días) a 37 °C. La evaluación se llevó a cabo con estereomicroscopios, equipo de radiografía digital con un software de análisis digital de imágenes y microscopía de polarización. Se formuló una integración de los índices de respuesta y se realizó un ANOVA. Resultados: Los hallazgos visuales, radiográficos e histológicos mostraron que en el G1 en los tiempos 1 a 3, la desmineralización se caracterizó por una gran pérdida de la integridad del esmalte (80 por ciento a 100 por ciento). Visualmente, el G2 a los 7 días mostró opacidad y pérdida de brillo (16 por ciento) con preservación de la estructura superficial del esmalte. Conclusiones: Se demuestra que el empleo de ácido láctico durante 7 días a pH 5,4 produce una lesión clínica, radiográfica e histológica similar a una lesión temprana del esmalte(AU)

Introduction: Abridged experimental methods are required to simulate early demineralizing lesions in a controlled and reproducible way. Objective: Perform an in vitro evaluation of a simple method of incipient enamel demineralization. Methods: Randomized experimental study with a double factorial replication design. Twelve third molars from healthy human subjects were selected for demineralization in a racemic lactic acid solution. Samples were then distributed randomly: Group 1 (G1) (n= 6) lactic acid at pH 2.4 and Group 2 (G2) (n= 6) lactic acid at pH 5.4. Each group was then subdivided (n = 2) to evaluate the effect of the solutions at three exposure times (7, 15 and 30 days) at 37°C. The evaluation used stereomicroscopes, a digital x-rays apparatus with software for the digital analysis of images, and polarization microscopy. An integration of the response indices was formulated and ANOVA was performed. Results: Visual, radiographic and histological findings showed that G1 at time 1 through 3 displayed demineralization characterized by extensive loss (80 percent to 100 percent) of enamel integrity. Visually, G2 at 7 days exhibited opacity and loss of brightness (16 percent), with preservation of the surface structure of the enamel. Conclusions: It was shown that employing lactic acid for 7 days at pH 5.4 develops a clinical, radiographic and histological injury similar to an early enamel lesion(AU)