Polarization enhancement mechanism from tissue staining in multispectral Mueller matrix microscopy.

Mueller matrix microscopy can provide comprehensive polarization-related optical and structural information of biomedical samples label-freely. Thus, it is regarded as an emerging powerful tool for pathological diagnosis. However, the staining dyes have different optical properties and staining mechanisms, which can put influence on Mueller matrix microscopic measurement. In this Letter, we quantitatively analyze the polarization enhancement mechanism from hematoxylin and eosin (H&E) staining in multispectral Mueller matrix microscopy. We examine the influence of hematoxylin and eosin dyes on Mueller matrix-derived polarization characteristics of fibrous tissue structures. Combined with Monte Carlo simulations, we explain how the dyes enhance diattenuation and linear retardance as the illumination wavelength changed. In addition, it is demonstrated that by choosing an appropriate incident wavelength, more visual Mueller matrix polarimetric information can be observed of the H&E stained tissue sample. The findings can lay the foundation for the future Mueller matrix-assisted digital pathology.

Characterization and classification of ductal carcinoma tissue using four channel based stokes-mueller polarimetry and machine learning.

Interaction of polarized light with healthy and abnormal regions of tissue reveals structural information associated with its pathological condition. Even a slight variation in structural alignment can induce a change in polarization property, which can play a crucial role in the early detection of abnormal tissue morphology. We propose a transmission-based Stokes-Mueller microscope for quantitative analysis of the microstructural properties of the tissue specimen. The Stokes-Mueller based polarization microscopy provides significant structural information of tissue through various polarization parameters such as degree of polarization (DOP), degree of linear polarization (DOLP), and degree of circular polarization (DOCP), anisotropy (r) and Mueller decomposition parameters such as diattenuation, retardance and depolarization. Further, by applying a suitable image processing technique such as Machine learning (ML) output images were analysed effectively. The support vector machine image classification model achieved 95.78% validation accuracy and 94.81% testing accuracy with polarization parameter dataset. The study's findings demonstrate the potential of Stokes-Mueller polarimetry in tissue characterization and diagnosis, providing a valuable tool for biomedical applications.

Stain-free enucleation of mouse and human oocytes with a 1033 nm femtosecond laser.

Significance: Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye. Aim: This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle. Approach: We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy. Results: We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization. Conclusions: This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.

Birefringence mapping of biological tissues based on polarization sensitive non-interferometric quantitative phase imaging technique.

OBJECTIVE: Oral cancer is a leading cause of mortality globally, particularly affecting developing regions where oral hygiene is often overlooked. The optical properties of tissues are vital for diagnostics, with polarization imaging emerging as a label-free, contrast-enhancing technique widely employed in medical and scientific research over past few decades. MATERIALS AND METHODS: We present a novel polarization sensitive quantitative phase imaging of biological tissues by incorporating the conventional polarization microscope and transport of intensity equation-based phase retrieval algorithm. This integration provides access to the birefringence mapping of biological tissues. The inherent optical anisotropy in biological tissues induces the polarization dependent refractive index variations which can provide the detailed insights into the birefringence characteristics of their extracellular constituents. Experimental investigations were conducted on both normal and cancerous oral tissue samples by recording a set of three polarization intensity images for each case with a step size of 2 µm. RESULTS: A noteworthy increment in birefringence quantification was observed in cancerous as compared to the normal tissues, attributed to the proliferation of abnormal cells during cancer progression. The mean birefringence values were calculated for both normal and cancerous tissues, revealing a significant increase in birefringence of cancerous tissues (2.1 ± 0.2) × 10-2 compared to normal tissues (0.8 ± 0.2) × 10-2. Data were collected from 8 patients in each group under identical experimental conditions. CONCLUSION: This polarization sensitive non-interferometric optical approach demonstrated effective discrimination between cancerous and normal tissues, with various parameters indicating elevated values in cancerous tissues.

Optical Sensor System for 3D Jones Matrix Reconstruction of Optical Anisotropy Maps of Self-Assembled Polycrystalline Soft Matter Films.

Our work uses a polarization matrix formalism to analyze and algorithmically represent optical anisotropy by open dehydration of blood plasma films. Analytical relations for Jones matrix reconstruction of optical birefringence maps of protein crystal networks of dehydrated biofluid films are found. A technique for 3D step-by-step measurement of the distributions of the elements of the Jones matrix or Jones matrix images (JMI) of the optically birefringent structure of blood plasma films (BPF) has been created. Correlation between JMI maps and corresponding birefringence images of dehydrated BPF and saliva films (SF) obtained from donors and prostate cancer patients was determined. Within the framework of statistical analysis of layer-by-layer optical birefringence maps, the parameters most sensitive to pathological changes in the structure of dehydrated films were found to be the central statistical moments of the 1st to 4th orders. We physically substantiated and experimentally determined the sensitivity of the method of 3D polarization scanning technique of BPF and SF preparations in the diagnosis of endometriosis of uterine tissue.

Analysis of polarization features of human breast cancer tissue by Mueller matrix visualization.

Significance: Breast cancer ranks second in the world in terms of the number of women diagnosed. Effective methods for its early-stage detection are critical for facilitating timely intervention and lowering the mortality rate. Aim: Polarimetry provides much useful information on the structural properties of breast cancer tissue samples and is a valuable diagnostic tool. The present study classifies human breast tissue samples as healthy or cancerous utilizing a surface-illuminated backscatter polarization imaging technique. Approach: The viability of the proposed approach is demonstrated using 95 breast tissue samples, including 35 healthy samples, 20 benign cancer samples, 20 grade-2 malignant samples, and 20 grade-3 malignant samples. Results: The observation results reveal that element m23 in the Mueller matrix of the healthy samples has a deeper color and greater intensity than that in the breast cancer samples. Conversely, element m32 shows a lighter color and reduced intensity. Finally, element m44 has a darker color in the healthy samples than in the cancer samples. The analysis of variance test results and frequency distribution histograms confirm that elements m23, m32, and m44 provide an effective means of detecting and classifying human breast tissue samples. Conclusions: Overall, the results indicate that surface-illuminated backscatter polarization imaging has significant potential as an assistive tool for breast cancer diagnosis and classification.

Multispectral polarization microscopy of different stages of human oral tissue: A polarization study.

Many optical techniques have been used in various diagnostics and biomedical applications since a decade and polarization imaging is one of the non-invasive and label free optical technique to investigate biological samples making it an important tool in diagnostics, biomedical applications. We report a multispectral polarization-based imaging of oral tissue by utilizing a polarization microscope system with a broadband-light source. Experiments were performed on oral tissue samples and multispectral Stokes mapping was done by recording a set of intensity images. Polarization-based parameters like degree of polarization, angle of fast axis, retardation and linear birefringence have been retrieved. The statistical moments of these polarization components have also been reported at multiples wavelengths. The polarimetric properties of oral tissue at different stages of cancer have been analyzed and significant changes from normal to pre-cancerous lesions to the cancerous are observed in linear birefringence quantification as (1.7 ± 0.1) × 10-3 , (2.5 ± 0.2) × 10-3 and (3.3 ± 0.2) × 10-3 respectively.

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.

Dependence of crack shape in loaded articular cartilage on the collagenous structure.

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages. METHODS: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance. RESULTS: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues. CONCLUSION: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.

Smartphone-based hand-held polarized light microscope for on-site pharmaceutical crystallinity characterization.

Polarized light microscopy (PLM) is a common but critical method for pharmaceutical crystallinity characterization, which has been widely introduced for research purposes or drug testing and is recommended by many pharmacopeias around the world. To date, crystallinity characterization of pharmaceutical solids is restricted to laboratories due to the relatively bulky design of the conventional PLM system, while little attention has been paid to on-site, portable, and low-cost applications. Herein, we developed a smartphone-based polarized microscope with an ultra-miniaturization design ("hand-held" scale) for these purposes. The compact system consists of an optical lens, two polarizers, and a tailor-made platform to hold the smartphone. Analytical performance parameters including resolution, imaging quality of interference color, and imaging reproducibility were measured. In a first approach, we illustrated the suitability of the device for pharmaceutical crystallinity characterization and obtained high-quality birefringence images comparable to a conventional PLM system, and we also showed the great promise of the device for on-site characterization with high flexibility. In a second approach, we employed the device as a proof of concept for a wider application ranging from liquid crystal to environmental pollutants or tissues from plants. As such, this smartphone-based hand-held polarized light microscope shows great potential in helping pharmacists both for research purposes and on-site drug testing, not to mention its broad application prospects in many other fields.

Single-shot Mueller-matrix imaging of zebrafish tissues: In vivo analysis of developmental and pathological features.

Zebrafish is a well-established animal model for developmental and disease studies. Its optical transparency at early developmental stages allows in vivo tissues visualization. Interaction of polarized light with these tissues provides information on their structure and properties. This approach is effective for muscle tissue analysis due to its birefringence. To enable real-time Mueller-matrix characterization of unanesthetized fish, we assembled a microscope for single-shot Mueller-matrix imaging. First, we performed a continuous observation of 48 species within the period of 2 to 96 hpf and measured temporal dependencies of the polarization features in different tissues. These measurements show that hatching was accompanied by a sharp change in the angle and degree of linearly polarized light after interaction with muscles. Second, we analyzed nine species with skeletal disorders and demonstrated that the spatial distribution of light depolarization features clearly indicated them. Obtained results demonstrated that real-time Mueller-matrix imaging is a powerful tool for label-free monitoring zebrafish embryos.

Location-Specific Study of Young Rabbit Femoral Cartilage by Quantitative µMRI and Polarized Light Microscopy.

OBJECTIVE: Microscopic magnetic resonance imaging (µMRI) and polarized light microscopy (PLM) are used to characterize the structural variations at different anatomical locations of femoral cartilage in young rabbits (12-14 weeks old). DESIGN: Four intact knees were imaged by µMRI at 86 µm resolution. Three small cartilage-bone specimens were harvested from each of 2 femoral medial condyles and imaged by quantitative µMRI (T2 anisotropy) at 9.75 µm resolution (N = 6). These specimens, as well as the other 2 intact femoral condyles, were used for histology and imaged by quantitative PLM (retardation and angle) at 0.25 µm to 4 µm resolutions. RESULTS: Quantitative MRI relaxation data and PLM fibril data revealed collaboratively distinct topographical variations in both cartilage thickness and its collagen organization in the juvenile joint. Cartilage characteristics from the central location have a 3-zone arcade-like fibril structure and a distinct magic angle effect, commonly seen in mature articular cartilage, while cartilage at the anterior location lacks these characteristics. Overall, the lowest retardation values and isotropic T2 values have been found in the distal femur (trochlear ridge), with predominant parallel fibers with respect to the articular surface. Central cartilage is the thickest (~550 µm), approximately twice as thick as the anterior and posterior locations. CONCLUSION: Distinctly different characteristics of tissue properties were found in cartilage at different topographical locations on femoral condyle in rabbits. Knowledge of location-specific structural differences in the collagen network over the joint surface can improve the understanding of local mechanobiology and provide insights to tissue engineering and degradation repairs.

Electric-field induced modulation of amorphous protein aggregates: polarization, deformation, and reorientation.

Proteins in their native state are only marginally stable and tend to aggregate. However, protein misfolding and condensation are often associated with undesired processes, such as pathogenesis, or unwanted properties, such as reduced biological activity, immunogenicity, or uncontrolled materials properties. Therefore, controlling protein aggregation is very important, but still a major challenge in various fields, including medicine, pharmacology, food processing, and materials science. Here, flexible, amorphous, micron-sized protein aggregates composed of lysozyme molecules reduced by dithiothreitol are used as a model system. The preformed amorphous protein aggregates are exposed to a weak alternating current electric field. Their field response is followed in situ by time-resolved polarized optical microscopy, revealing field-induced deformation, reorientation and enhanced polarization as well as the disintegration of large clusters of aggregates. Small-angle dynamic light scattering was applied to probe the collective microscopic dynamics of amorphous aggregate suspensions. Field-enhanced local oscillations of the intensity auto-correlation function are observed and related to two distinguishable elastic moduli. Our results validate the prospects of electric fields for controlling protein aggregation processes.

Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy.

Current tools lack the temporal or spatial resolution necessary to image many important aspects of the architecture and dynamics of the optic nerve head (ONH). We evaluated the potential of instant polarized light microscopy (IPOL) to overcome these limitations by leveraging the ability to capture collagen fiber orientation and density in a single image. Coronal sections through the ONH of fresh normal sheep eyes were imaged using IPOL while they were stretched using custom uniaxial or biaxial micro-stretch devices. IPOL allows identifying ONH collagen architectural details, such as fiber interweaving and crimp, and has high temporal resolution, limited only by the frame rate of the camera. Local collagen fiber orientations and deformations were quantified using color analysis and image tracking techniques. We quantified stretch-induced collagen uncrimping of lamina cribrosa (LC) and peripapillary sclera (PPS), and changes in LC pore size (area) and shape (convexity and aspect ratio). The simultaneous high spatial and temporal resolutions of IPOL revealed complex ONH biomechanics: i) stretch-induced local deformation of the PPS was nonlinear and nonaffine. ii) under load the crimped collagen fibers in the PPS and LC straightened, without torsion and with only small rotations. iii) stretch-induced LC pore deformation was anisotropic and heterogeneous among pores. Overall, with stretch the pores were became larger, more convex, and more circular. We have demonstrated that IPOL reveals details of collagen morphology and mechanics under dynamic loading previously out of reach. IPOL can detect stretch-induced collagen uncrimping and other elements of the tissue nonlinear mechanical behavior. IPOL showed changes in pore morphology and collagen architecture that will help improve understanding of how LC tissue responds to load.

Structural differences between immature and mature articular cartilage of rabbits by microscopic MRI and polarized light microscopy.

This study aimed to determine the structural features between immature and mature articular cartilage from the humeral and femoral joints of rabbits. Specimens of articular cartilage (n = 6 for immature tissue, n = 6 for mature tissue) that were still attached to the underlying bone from a humerus (shoulder joint) or femur (knee joint) were imaged using microscopic MRI (µMRI) and polarized light microscopy (PLM). Quantitative µMRI data with a pixel resolution of 11.7-13.2 µm revealed a number of differences between the immature and mature cartilage, including total thickness, and T2 and T1ρ relaxation values. Quantitative PLM data with a pixel resolution of 0.25-1 µm confirmed the µMRI results and revealed additional differences in cellular features between the tissues. The mature cartilage had a clearly defined tidemark, which was absent in the immature tissue. The ability to differentiate specific maturation-related cartilage characteristics could be beneficial to translational studies of degenerative diseases such as osteoarthritis.

Computational image translation from Mueller matrix polarimetry to bright-field microscopy.

Mueller matrix (MM) polarimetry can provide comprehensive information about the polarization properties that are closely related to the microstructural features and has demonstrated its potential in biomedical studies and clinical practices, and bright-field microscopy is widely used in pathological diagnosis as the golden standard. In this work, we improve the throughput of MM microscopy by learning a statistical transformation between these two imaging systems based on deep learning. Using this approach, the MM microscope can generate an image that is equivalent to a bright-field microscope image of the matching field of view. We add new transformative capability to the existing MM imaging system without requiring extra hardware. The translation model is based on conditional generative adversarial network with customized loss functions. We demonstrated the effectiveness of our approach on liver and breast tissues and evaluated the performance by four quantitative similarity assessment methods in pixel, image and distribution levels, respectively.

Full-Range Optical Imaging of Planar Collagen Fiber Orientation Using Polarized Light Microscopy.

A novel method for semiautomated assessment of directions of collagen fibers in soft tissues using histological image analysis is presented. It is based on multiple rotated images obtained via polarized light microscopy without any additional components, i.e., with just two polarizers being either perpendicular or nonperpendicular (rotated). This arrangement breaks the limitation of 90° periodicity of polarized light intensity and evaluates the in-plane fiber orientation over the whole 180° range accurately and quickly. After having verified the method, we used histological specimens of porcine Achilles tendon and aorta to validate the proposed algorithm and to lower the number of rotated images needed for evaluation. Our algorithm is capable to analyze 5·105 pixels in one micrograph in a few seconds and is thus a powerful and cheap tool promising a broad application in detection of collagen fiber distribution in soft tissues.

Angular distribution of luminescence dissymmetry observed from a random laser built upon the exocuticle of the scarab beetle Chrysina gloriosa.

We investigate the angular distribution of luminescence dissymmetry of random lasing in the mixture of rhodamine 6G and titanium dioxide nanoparticles upon a biocompatible natural material substrate, i.e., the elytron of the scarab beetle Chrysina gloriosa. We look into both green and gold-colored areas of the elytron that exhibit distinctly different circular dichroism properties. The fabricated sample asymmetrically emits both left- and right-handed circularly polarized light at 570 nm when pumped at 532 nm, depending on the direction of emission and the angle of the pump incidence. We characterize the light via measuring the angular distribution of its luminescence dissymmetry factor (g lum), which reaches an unusually high maximal value of 0.90 or -0.50 at some specific angle depending on the handedness of its polarization. This random laser source can be used in numerous potential optoelectronic applications which require light emission of distributed luminescence dissymmetry or of high luminescence dissymmetry.

Real-time polarization microscopy of fibrillar collagen in histopathology.

Over the past two decades, fibrillar collagen reorganization parameters such as the amount of collagen deposition, fiber angle and alignment have been widely explored in numerous studies. These parameters are now widely accepted as stromal biomarkers and linked to disease progression and survival time in several cancer types. Despite all these advances, there has not been a significant effort to make it possible for clinicians to explore these biomarkers without adding steps to the clinical workflow or by requiring high-cost imaging systems. In this paper, we evaluate previously described polychromatic polarization microscope (PPM) to visualize collagen fibers with an optically generated color representation of fiber orientation and alignment when inspecting the sample by a regular microscope with minor modifications. This system does not require stained slides, but is compatible with histological stains such as H&E. Consequently, it can be easily accommodated as part of regular pathology review of tissue slides, while providing clinically useful insight into stromal composition.

Differential Polarization Imaging of Plant Cells. Mapping the Anisotropy of Cell Walls and Chloroplasts.

Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general-in addition to their high sensitivity, fast data acquisition, and great versatility of 2D-4D image analyses-also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments-making them capable of measuring different polarization spectroscopy parameters, parallel with the 'conventional' intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies.