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Recently, virtual staining techniques have attracted more and more attention, which can help bypass the chemical staining process of traditional histopathological examination, saving time and resources. Meanwhile, as an emerging tool to characterize specific tissue structures in a label-free manner, the Mueller matrix microscopy can supplement more structural information that may not be apparent in bright-field images. In this Letter, we propose the Mueller matrix guided generative adversarial networks (MMG-GAN). By integrating polarization information provided by the Mueller matrix microscopy, the MMG-GAN enables the effective transformation of input H&E-stained images into corresponding Masson trichrome (MT)-stained images. The experimental results demonstrate the accuracy of the generated images by MMG-GAN and reveal the potential for more stain transformation tasks by incorporating the Mueller matrix polarization information, laying the foundation for future polarimetry-assisted digital pathology.

Systemic amyloidosis involves the deposition of misfolded proteins in organs/tissues, leading to progressive organ dysfunction and failure. Congo red is the gold-standard chemical stain for visualizing amyloid deposits in tissue, showing birefringence under polarization microscopy. However, Congo red staining is tedious and costly to perform, and prone to false diagnoses due to variations in amyloid amount, staining quality and manual examination of tissue under a polarization microscope. We report virtual birefringence imaging and virtual Congo red staining of label-free human tissue to show that a single neural network can transform autofluorescence images of label-free tissue into brightfield and polarized microscopy images, matching their histochemically stained versions. Blind testing with quantitative metrics and pathologist evaluations on cardiac tissue showed that our virtually stained polarization and brightfield images highlight amyloid patterns in a consistent manner, mitigating challenges due to variations in chemical staining quality and manual imaging processes in the clinical workflow., (© 2024. The Author(s).)

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.

As bones age in most mammals, they typically become more fragile. This state of bone fragility is often associated with more homogenous collagen fiber orientations (CFO). Unlike most mammals, bats maintain mechanically competent bone throughout their lifespans, but little is known of positional and age-related changes in CFO within wing bones. This study tests the hypothesis that age-related changes in CFO in big brown bats (Eptesicus fuscus) differ from those of the standard mammalian model for skeletal aging, the C57BL/6 laboratory mouse. We used data from quantitative polarized light microscopy (qPLM) to compare CFO across the lifespan of long-lived big brown bats and age matched C57BL/6 mice. Eptesicus and C57BL/6 mice displayed idiosyncratic patterns of CFO. Consistent age-related changes were only apparent in the outer cortical bone of Eptesicus, where bone tissue is more longitudinally arranged and more anisotropic in older individuals. Both taxa displayed a ring of more transversely oriented bone tissue surrounding the medullary cavity. In Eptesicus, this tissue represents a greater proportion of the overall cross-section, and is more clearly helically aligned (arranged at 45° to the bone long axis) than similar bone tissue in mice. Bat wing bones displayed a proximodistal gradient in CFO anisotropy and longitudinal orientation in both outer and inner cortical bone compartments. This study lays a methodological foundation for the quantitative evaluation of bone tissue architecture in volant and non-volant mammals that may be expanded in the future., (© 2023 The Authors. The Anatomical Record published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

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., (© 2024 The Authors.)

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., (© 2024. The Author(s).)

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 m 23 in the Mueller matrix of the healthy samples has a deeper color and greater intensity than that in the breast cancer samples. Conversely, element m 32 shows a lighter color and reduced intensity. Finally, element m 44 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 m 23 , m 32 , and m 44 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., (© 2024 The Authors.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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.

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., (© 2023 Wiley-VCH GmbH.)

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., (© 2023 The Authors.)

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., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (© 2022 Wiley-VCH GmbH.)

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., (© 2022 Anatomical Society.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2021 Wiley-VCH GmbH.)

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., (© 2022. The Author(s).)

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.

Objective: In order to appreciate the roles articular cartilage of sesamoid bones and sesamoid fibrocartilage play in anatomy and pathology, the articular cartilage of the patella ( n = 4) and suprapatella ( n = 4) (a sesamoid fibrocartilage) of 12 to 14 weeks old New Zealand rabbits were studied qualitatively and quantitatively., Design/method: The intact knee joints and block specimens from the joints were imaged using microscopic magnetic resonance imaging (µMRI) at a 97.6-µm pixel resolution for the former and 19.5-µm resolution for the latter. Histological sections were made out of the µMRI-imaged specimens, which were imaged using polarized light microscopy (PLM) at 0.25-, 1-, and 4-µm pixel resolutions., Results: The patella cartilage varied in thickness across the medial to lateral ends of the sesamoid bone with the central medial aspect slightly thicker than the lateral aspect. The suprapatella fibrocartilage decreased proximally away from the knee joint. Quantitative results of patellar cartilage showed strong dependence of fiber orientation with the tissue depth. Three histological zones can be clearly observed, which are similar to articular cartilage from other large animals. The sesamoid fibrocartilage has one thin surface layer (10 µm thick) of parallel-arranged structured fibers followed immediately by the majority of random fibers in bulk tissue. T2 relaxation time anisotropy was observed in the patellar cartilage but not in the bulk fibrocartilage., Conclusion: Given the different functions of these 2 different types of cartilages in joint motion, these quantitative results will be beneficial to future studies of joint diseases using rabbits as the animal model.

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 5·10 5 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., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Michaela Turčanová et al.)

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.

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., (© 2021. The Author(s).)

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.

A new, to the best of our knowledge, method for Stokes vector imaging is proposed to achieve imaging and dynamic monitoring of a non-labeled cytomembrane. In this work, a polarization state vector is described by a Stokes vector and expressed in chrominance space. A physical quantity called polarization chromaticity value (PCV) corresponding to a Stokes vector is used as the imaging parameter to perform Stokes vector imaging. By using the PCV imaging technique, the Stokes vector can be expressed in three-dimensional real space rather than in a Poincare sphere. Furthermore, a four-way Stokes parameter confocal microscopy system is designed to measure four Stokes parameters simultaneously and obtain micro-imaging. Label-free living onion cell membranes and their plasmolysis process are selected as the representative micro-anisotropy experimental analysis. It is proved that PCV imaging can perform visualization of cytomembranes, and further, microscopic orientation is demonstrated. The prospect of universal measurement of anisotropy details for analysis and diagnosis is provided.

The ratio of type III to type I collagen is important for properly maintaining functions of organs and cells. We propose a method to quantify the ratio of type III to total (type I + III) collagen (λ III ) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the λ III of collagen gels was examined, and the slope (k 1 ) and SHG light intensity at 0% type III collagen (k 2 ) were determined. Second, the SHG light intensity of a 100% type I collagen fiber bundle and its diameter (D) were measured, and the slope (k 3 ) of the relationship was determined. The λ III in a collagen fiber bundle was estimated from these constants (k 1-3 ) and SHG light intensity. We applied this method to collagen fiber bundles isolated from the media and adventitia of porcine thoracic aortas, and obtained λ III  = 84.7% ± 13.8% and λ III  = 17.5% ± 15.2%, respectively. These values concurred with those obtained with a typical quantification method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings demonstrated that the method proposed is useful to quantify the ratio of type III to total collagen in a collagen fiber bundle.

This work presents the results of in vivo measurements of human corneal birefringence properties using the one-way reflective polariscope, in which the same module is used to generate and analyze the light polarization state. Data analysis was performed with the use of Mueller polarimetry techniques. The distribution of the azimuth angle and the phase retardation was determined on the paracentral and limbal areas. The results indicate growth of the phase retardation magnitude and the radial orientation of the azimuth angle in the cornea's peripheral region, which confirms our assumptions and the results presented by other researchers.

Flavonoid compounds are known for their antibacterial, anti-inflammatory, and anticancer properties. Therefore, they can influence membrane properties that interest us, modifying both their structure and functions. We used kaempferol (K) and myricetin (M) as representatives of this group. We investigated the influence of the abovementioned compounds on model cell membranes' properties (i.e., Langmuir monolayers and liposomes). The basic research methods used in these studies were the Langmuir method with Brewster angle microscopy and microelectrophoresis. The π-A isotherms were registered for the pure components and mixtures of these compounds with phosphatidylcholine (PC) in appropriate volume ratios. Using mathematical equations, we established that kaempferol, myricetin, and the lipids formed complexes at 1:1 ratios. We derived the parameters characterizing the formed complexes, i.e., the surfaces occupied by the complexes and the stability constants of the formed complexes. Using the microelectrophoretic method, we determined the dependence of the lipid membranes' surface charge density as a function of the pH (in the range of 2 to 10) of the electrolyte solution. The presented results indicate that the PC membrane's modification with kaempferol or myricetin affected changes in the surface charge density and isoelectric point values., Competing Interests: The authors declare no conflict of interest.

Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.

Intranuclear birefringent inclusions (IBI) found in various cell types in paraffin-embedded tissue sections have long been considered to be a tissue processing artifact, although an association with biological processes has been suggested. We applied polychromatic polarization microscopy to image their spatial organization. Our study provides evidence that IBI are caused by liquid paraffin-macromolecular crystals formed during paraffin-embedding procedures within cells and potentially reflect an active transcriptional status.

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.

Mueller matrix (MM) imaging has demonstrated its potential application in much research, especially in probing delicate and complex biomedical specimens. Qualities of MM images are important for further quantitative characterization. In this paper, we compare the performance and imaging qualities of three calibration methods. Air, waveplate and cell specimen are selected as standard samples for comparison. In addition, we also propose two general MM imaging quality indices that can be used as quantitative evaluations for MM imaging systems and calculation processes based on real samples. The numerical calibration method turns out to give the best accuracy and precision, as well as the best image qualities.

Optical interrogation of tissues is broadly considered in biomedical applications. Nevertheless, light scattering by tissue limits the resolution and accuracy achieved when investigating sub-surface tissue features. Light carrying optical angular momentum or complex polarization profiles, offers different propagation characteristics through scattering media compared to light with unstructured beam profiles. Here we discuss the behaviour of structured light scattered by tissue-mimicking phantoms. We study the spatial and the polarization profile of the scattered modes as a function of a range of optical parameters of the phantoms, with varying scattering and absorption coefficients and of different lengths. These results show the non-trivial trade-off between the advantages of structured light profiles and mode broadening, stimulating further investigations in this direction.

Tissue birefringence is an intrinsic marker of potential value for cancer diagnosis. Traditionally, birefringence properties have been studied by using intensity-based formalisms, through the Mueller matrix algebra. On the other hand, the Jones matrix description allows for a direct assessment of the sample's anisotropic response. However, because Jones algebra is based on complex fields, requiring measurements of both phase and amplitude, it is less commonly used. Here we propose a real-time imaging method for measuring Jones matrices by quantitative phase imaging. We combine a broadband phase imaging system with a polarization-sensitive detector to obtain Jones matrices at each point in a megapixel scale image, with near video rate capture speeds. To validate the utility of our approach, we measured standard targets, partially birefringent samples, dynamic specimens, and thinly sliced histopathological tissue.

In this paper, we used a convolutional neural network to study the classification of marine microalgae by using low-resolution Mueller matrix images. Mueller matrix images of 12 species of algae from 5 families were measured by a Mueller matrix microscopy with an LED light source at 514 nm wavelength. The data sets of seven resolution levels were generated by the bicubic interpolation algorithm. We conducted two groups of classification experiments; one group classified the algae into 12 classes according to species category, and the other group classified the algae into 5 classes according to family category. In each group of classification experiments, we compared the classification results of the Mueller matrix images with those of the first element (M11) images. The classification accuracy of Mueller matrix images declines gently with the decrease of image resolution, while the accuracy of M11 images declines sharply. The classification accuracy of Mueller matrix images is higher than that of M11 images at each resolution level. At the lowest resolution level, the accuracy of 12-class classification and 5-class classification of full Mueller matrix images is 29.89% and 35.83% higher than those of M11 images, respectively. In addition, we also found that the polarization information of different species had different contributions to the classification. These results show that the polarization information can greatly improve the classification accuracy of low-resolution microalgal images.

The growing use of therapeutic proteins requires accurate analytical techniques for measuring biophysical and structural changes during manufacturing. This is particularly true for the PEGylation of proteins, because characterization of PEGylation reactions and products can often be difficult due to the relatively small impact on protein structure, the lack of an accessible polyethylene glycol (PEG) chromophore, and the heterogeneous final product mixtures. Intrinsic fluorescence spectroscopy is one potential solution due to its relatively high sensitivity to small changes in protein structure and its suitability for online or atline measurements. In this study, we use the PEGylation of lysozyme as a model system to determine the efficacy of polarized excitation-emission matrix (pEEM) spectroscopy as a rapid tool for characterizing the structural variability of the lysozyme (LZ) starting materials and PEGylated products with varying PEG-to-protein ratios (PPR). Dynamic light scattering showed that as PPR increased from 0 to 2.8, the hydrodynamic radius increased from ∼2.2 to 4.8 nm. pEEM measurements provided several sources of information: Rayleigh scattering to identify size changes and aggregate/particle formation, and fluorescence emission to assess chemical and structural changes. PEGylation induced sufficient physicochemical changes in LZ, which produced changes in the pEEM spectra, largely due to variations in the hydrophobic environments of tryptophan residues close to a PEG attachment site. These significant spectral changes when modeled using conventional multivariate analysis methods were able to easily discriminate the raw product solutions according to the degree of PEGylation and were also able to predict PPR with reasonable accuracy (root mean square error for calibration ∼10%, relative error of prediction < 20%), considering the reference size exclusion chromatography method error of ∼7.2%. The variable selection of the pEEM data suggests that equivalent predictions could be obtained with faster and simpler two-dimensional spectra, making the method a more viable online measurement method., (© 2020 Wiley Periodicals LLC.)

Vaterite helicoids [W. Jiang et al., Nat. Commun., 2017, 8, 15066] are chiral, polycrystalline suprastructures grown in the presence of the amino acids, aspartic (Asp) or glutamic (Glu) acid, that are abundant in proteins regulating biomineralization. These complex objects are composed of hexagonal vaterite nanocrystals assembled as curved-edge platelets that form chiral ensembles. The sense stacked platelets is correlated with the stereochemistry of the amino acid additive: l-Asp gives counterclockwise architectures while d-Asp gives the clockwise enantiomorphs. As new layers stack, platelets become progressively inclined with respect to the substrate suface. The growth and structure of vaterite helicoids was originally evidenced by electron microscopy and atomic force microscopy. Here, we develop an optical model for describing polarized light transmission through helicoids as measured by Mueller matrix polarimetry. The close agreement between experimental measurements and simulation confirms that the propellor-like organization of inclined platelets creates optically active structures determined by growth additive stereochemistry. The microscopy employed demonstrates the information that can be obtained by complete polarimetry using a camera as a light detector, a technique that could be applied profitably to all manner of complex structures organized from anisotropic particles.

Time-resolved Mueller matrix (MM) imaging polarimetry in transmission mode has been implemented in both epithelium and stromal regions of cervical tissues to explore the various polarization dynamics in connection with the diagnosis of cervical precancer. The picosecond-resolved intensity patterns of various MM elements, resulting from the various orders of scattering, at different time delays provide clear demarcation between the epithelium and stroma of cervical tissue. The time dependent depolarization and retardance maps are seen to differentiate the epithelium from stroma. The average values of time dependent linear, linear-45, and circular depolarization and linear, circular, and scalar retardance parameters in different regimes of scattering from the optically anisotropic stromal region identify the pre-malignancy in cervical tissue. As the disease evolves, time dependent linear depolarization varies to larger values as compared to time dependent circular depolarization. Interestingly, the chirality of the collagen network that rotates the plane of polarized light in either direction in normal samples is limited to only the clockwise direction during the progression of the disease. These results show potential in the early detection and understanding of the mechanisms of morphological changes in cervical cancer development.

New seven-ring systems of dipyridine derivative liquid crystalline 2:1 supramolecular H-bonded complexes were formed between 4-n-alkoxyphenylazo benzoic acids and 4-(2-(pyridin-4-yl)diazenyl)phenyl nicotinate. Mesomorphic behaviors of the prepared complexes were investigated using a combination of differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). Fermi bands attributed to the presence of intermolecular H-bond interactions were confirmed by FT-IR spectroscopy. All prepared complexes possessed an enantiotropic nematic phase with a broad temperature nematogenic range. Phases were confirmed by miscibility with a standard nematic (N) compound. A comparison was constructed to investigate the influence of the incorporation of the azophenyl moiety on the mesomeric behavior of corresponding five-membered complexes. It was found that the present complexes observed induced a wide nematic phase with relatively higher temperature ranges than the five aromatic systems. Density functional theory (DFT ) suggested the nonlinear geometry of the formed complex. The results of the DFT explained the nematic mesophase formation. Moreover, the π-π stacking of the aromatic moiety in the phenylazo acid plays an effective role in the mesomorphic thermal stability. The energy difference between the frontier molecular orbitals, HOMO (highest occupied) and LUMO (lowest occupied), and the molecular electrostatic potential (MEP) of the prepared complexes were estimated by DFT calculations. The results were used to illustrate the observed nematic phase for all H-bonded supramolecular complexes. Finally, photophysical studies were discussed which were carried out by UV spectroscopy connected to a hot stage., Competing Interests: The authors declare no conflicts of interest.

Physiological states of marine microalgal cells can influence photosynthesis efficiency, which affects approximately half of global carbon fixation. The detection of the algae physiological profiles is important for marine ecology and economy. In this paper, we propose a polarized light-scattering method to detect sensitive changes in the physiological states of the suspended marine microalgal cells. Our experimental setup is designed to measure the scattered polarization parameters of the cells suspended individually in the seawater. Two species of microalgal cells cultured in the laboratory were measured for several days. Experimental results showed that both species display distinctive changes in their polarized photon scattering features corresponding to changes in their physiological states. The changes are far more prominent than those displayed in unpolarized light scattering. Microscopy observations, simulations for microspheres of different diameters and refractive indices, or different shapes, indicated that the polarization features of the scattered photons are sensitive to the submicrometer microstructures of the cells. This study demonstrates the potential of the polarized light-scattering technique to characterize the physiological states of suspended marine microalgae.

Analysis of spatial frequency of Mueller matrix (MM) images in the Fourier domain yields quantifying parameters of anisotropy in the stromal region in normal and precancerous tissue sections of human uterine cervix. The spatial frequencies of MM elements reveal reliable information of microscopic structural organization arising from the different orientations of collagen fibers in the connective tissue and their randomization with disease progression. Specifically, the local disorder generated in the normal periodic and regular structure of collagen during the growth of the cervical cancer finds characteristic manifestation in the Fourier spectrum of the selected Mueller matrix elements encoding the anisotropy effects through retardance and birefringence. In contrast, Fourier spectra of differential polarization gated images are limited to only one orientation of collagen. Fourier spectra of first row elements M11, M12, M13, and M14 and first column elements M11, M21, M31, and M41 discriminates cervical inter-epithelial neoplasia (CIN)-I from normal cervical tissue samples with 95%-100% sensitivity and specificity. FFT spectra of first and fourth row elements classify CIN-I and CIN-II grades of cervical cancerous tissues with 90%-100% sensitivity and 87%-100% specificity. Normal and CIN-II grade samples are successfully discriminated through Fourier spectra of every MM element while that of M31 element arises as the key classifier among normal, CIN-I, and CIN-II grades of cervical cancer with 100% sensitivity and specificity. These results demonstrate the promise of spatial frequency analysis of Mueller matrix images as a novel, to the best of our knowledge, approach for cancer/precancer detection.

We develop a novel label-free liquid crystal (LC) aptasensor based on intrinsic properties of nematic LCs for ultra-sensitive detection of tetracycline. The aptasensor is assembled by immobilizing aptamers onto the glass slide modified with both homeotropic alignment and silane coupling agents. Designed aptasensor makes use of the target-induced aptamer conformational switching and disruption of the orientation of LCs which lead to an obvious change of the optical appearance from a dark to a bright response. We describe the optimized condition for maintaining the homeotropic orientation of LCs, which are suitable for the tetracycline detection. The average gray-scale intensities of polarizing optical microscopy images were calculated to quantitatively detect tetracycline concentrations. The aptasensor works especially at trace level of tetracycline as low as 0.5 pM. Moreover, the LC aptasensor was successfully used to detect tetracycline in the real milk sample. According to the results, the proposed LC aptasensor for tetracycline detection is simple, ultra-sensitive, label free and ease of preparation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Jamin-Lebedeff (JL) polarization interference microscopy is a classical method for determining the change in the optical path of transparent tissues. Whilst a differential interference contrast (DIC) microscopy interferes an image with itself shifted by half a point spread function, the shear between the object and reference image in a JL-microscope is about half the field of view. The optical path difference (OPD) between the sample and reference region (assumed to be empty) is encoded into a color by white-light interference. From a color-table, the Michel-Levy chart, the OPD can be deduced. In cytology JL-imaging can be used as a way to determine the OPD which closely corresponds to the dry mass per area of cells in a single image. Like in other interference microscopy methods (e.g. holography), we present a phase retrieval method relying on single-shot measurements only, thus allowing real-time quantitative phase measurements. This is achieved by adding several customized 3D-printed parts (e.g. rotational polarization-filter holders) and a modern cellphone with an RGB-camera to the Jamin-Lebedeff setup, thus bringing an old microscope back to life. The algorithm is calibrated using a reference image of a known phase object (e.g. optical fiber). A gradient-descent based inverse problem generates an inverse look-up-table (LUT) which is used to convert the measured RGB signal of a phase-sample into an OPD. To account for possible ambiguities in the phase-map or phase-unwrapping artifacts we introduce a total-variation based regularization. We present results from fixed and living biological samples as well as reference samples for comparison., Competing Interests: The authors have declared that no competing interests exist.

Zebrafish are powerful animal models for understanding biological processes and the molecular mechanisms involved in different human diseases. Advanced optical techniques based on fluorescence microscopy have become the main imaging method to characterize the development of these organisms at the microscopic level. However, the need for fluorescence probes and the consequent high light doses required to excite fluorophores can affect the biological process under observation including modification of metabolic function or phototoxicity. Here, without using any labels, we propose an implementation of a Mueller-matrix polarimeter into a commercial optical scanning microscope to characterize the polarimetric transformation of zebrafish preserved at different embryonic developmental stages. By combining the full polarimetric measurements with statistical analysis of the Lu and Chipman mathematical decomposition, we demonstrate that it is possible to quantify the structural changes of the biological organization of fixed zebrafish embryos and larvae at the cellular scale. This convenient implementation, with low light intensity requirement and cheap price, coupled with the quantitative nature of Mueller-matrix formalism, can pave the way for a better understanding of developmental biology, in which label-free techniques become a standard tool to study organisms.

We introduce an iterative method for designing optical phantoms that are able to replicate the depolarization profiles of various target media, including colloidal suspensions of Intralipid, bovine milk, and ex vivo samples of ovine kidney cortex tissue. The designed phantoms comprise spherical scattering particles with fine-tuned size distributions and are capable of simultaneously reproducing spatially resolved intensity measurements and depolarization measurements of target media when illuminated with circularly polarized light.

Collagen fiber organization requires characterization in many biomedical applications, but it is difficult to objectively quantify in standard histology tissue sections. Quantitative polarized light imaging is a low-cost technique that allows for rapid measurement of collagen fiber orientation and thickness. In this study, we utilize a quantitative polarized light imaging system to characterize fiber orientation and thickness from wound sections. Full thickness skin wound sections that were previously stained with hematoxylin and eosin were used to assess collagen fiber content and organization at different points during the wound healing process. Overall, wounds exhibited a measurable increase in collagen fiber thickness and a nonlinear change in fiber reorganization within the wound. Our study demonstrates that quantitative polarized light imaging is an inexpensive alternative or supplement to standard histology protocols, requiring no additional stains or dyes, and yields repeatable quantitative assessments of collagen organization., (© 2019 by the Wound Healing Society.)

Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

Background: Oral submucous fibrosis (OSMF), a well-recognized oral potentially malignant disorder, results due to increased collagen production and reduced collagen degradation., Aims and Objectives: To qualitatively compare the staining properties of collagen in OSMF using two special stains based on their birefringent property using polarizing microscopy. The study also assessed the distribution and orientation of collagen fibers in different grades of OSMF., Materials and Methods: A total of 73 subjects with different clinical and histopathological staging of OSMF comprised the study population. Histopathological examination was done using hematoxylin and eosin stain, Van Gieson and picrosirius red. Collagen fibers were analyzed for polarization colors, distribution, and orientation., Results: Picrosirius red stained both thick and thin collagen fibers. Irrespective of the histopathological grades reddish orange and yellowish orange were the most predominant colors. Parallel arrangement of fibers was observed when stained with Van Gieson but picrosirius red stained sections showed a majority of parallel type I fibers with perpendicular type III fibers which increased with advancement in the histopathological grade. Yellowish orange and greenish yellow fibers were predominant in the lamina propria, while reddish orange fibers were predominant in the submucosa., Conclusion: Picrosirius red was found to be a better stain. Histopathological grading and polarization colors showed no association with each other. Collagen fibers were more thickly and tightly packed in the submucosa indicating that the process of fibrosis began there. The increase in perpendicular type III fibers with advancing histopathological grades suggested their role in fibrosis.

Detection of biomarkers in body fluids is critical to both diagnosing the life-threatening diseases and optimizing therapeutic interventions. We herein report use of liquid crystals (LCs) to detect biomarkers in blood with high sensitivity and specificity by employing in situ rolling circle amplification (RCA) on magnetic beads (MBs). Specific recognition of cancer biomarkers, such as platelet derived growth factor BB (PDGF-BB) and adenosine, by aptamers leads to formation of a nucleic acid circle on MBs preassembled with ligation DNA, linear padlock DNA, and aptamers, thereby triggering in situ RCA. LCs change from dark to bright appearance after the in situ RCA products being transferred onto the LC interface decorated with octadecy trimethylammonium bromide (OTAB), which is particularly sensitive to the amplified DNA on MBs. Overall, this label-free approach takes advantages of high specificity of aptamer-based assay, efficient enrichment of signaling molecules on MBs, remarkable DNA elongation performance of the RCA reaction, and high sensitivity of LC-based assay. It successfully eliminates the matrix interference on the LC-based sensors and thus achieves at least 4 orders of magnitude improvement in sensitivity for detection of biomarkers compared to other LC-based sensors. In addition, performance of the developed sensor is comparable to that of the commercial ones. Thus, this study provides a simple, powerful, and promising approach to facilitate highly sensitive, specific, and label-free detection of biomarkers in body fluids.