Birefringence-induced phase delay enables Brillouin mechanical imaging in turbid media.

Acoustic vibrations of matter convey fundamental viscoelastic information that can be optically retrieved by hyperfine spectral analysis of the inelastic Brillouin scattered light. Increasing evidence of the central role of the viscoelastic properties in biological processes has stimulated the rise of non-contact Brillouin microscopy, yet this method faces challenges in turbid samples due to overwhelming elastic background light. Here, we introduce a common-path Birefringence-Induced Phase Delay (BIPD) filter to disentangle the polarization states of the Brillouin and Rayleigh signals, enabling the rejection of the background light using a polarizer. We demonstrate a 65 dB extinction ratio in a single optical pass collecting Brillouin spectra in extremely scattering environments and across highly reflective interfaces. We further employ the BIPD filter to image bone tissues from a mouse model of osteopetrosis, highlighting altered biomechanical properties compared to the healthy control. Results herald new opportunities in mechanobiology where turbid biological samples remain poorly characterized.

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.

An integrated model of the human cornea as a linear biaxial birefringent medium.

A novel model of human corneal birefringence is presented. The cornea is treated as a homogeneous biaxial linear birefringent medium in which the values of the binormal axes angle and organization of the main refractive indices vary continuously from the apex to the limbus. In its central part, the angle between binormal axes is 35°, and para centrally, it smoothly increases to 83.7°. The values of the main refractive indices (nx, ny, nz) change, as well as their order, from nx < nz < ny to nz < nx < ny. The transition between these two states was described with a normal distribution (µ = 0.45, σ = 0.1). The presented model corresponds with the experimental results presented in the literature. To our knowledge, it is the first model that presents the anisotropic properties' distributions of the entire cornea. The presented model facilitates a better understanding of the corneal birefringence phenomenon directly related to its lamellar structure.

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.

Study on anisotropy orientation due to well-ordered fibrous biological microstructures.

Significance: Most biological fibrous tissues have anisotropic optical characteristics, which originate from scattering by their fibrous microstructures and birefringence of biological macromolecules. The orientation-related anisotropic interpretation is of great value in biological tissue characterization and pathological diagnosis. Aim: We focus on intrinsic birefringence and form birefringence in biological tissue samples. By observing and comparing the forward Mueller matrix of typical samples, we can understand the interpretation ability of orientation-related polarization parameters and further distinguish the sources and trends of anisotropy in tissues. Approach: For glass fiber, silk fiber, skeletal muscle, and tendon, we construct a forward measuring device to obtain the Mueller matrix image and calculate the anisotropic parameters related to orientation. The statistical analysis method based on polar coordinates can effectively analyze the difference in anisotropic parameters. Results: For those birefringent fibers, the statistical distribution of fast-axis values derived from Mueller matrix polar decomposition was found to exhibit bimodal characteristics, which is a key point in distinguishing the single-layer birefringent fiber sample from a layered, multioriented fibrous sample. The application conditions and interference factors of anisotropic orientation parameters are analyzed. Based on the parameters extracted from the orientation bimodal distribution, we can evaluate the relative change trend of intrinsic birefringence and form birefringence in anisotropic samples. Conclusions: The cross-vertical bimodal distribution of the fast axis of anisotropic fibers is beneficial to accurately analyze the anisotropic changes in biological tissues. The results imply the potential of anisotropic orientation analysis for applications in pathological diagnosis.

Acetylation of homogalacturonan and rhamnogalacturonan-I is catalyzed by a suite of trichome birefringence-like proteins.

Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about the O-acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl-CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan-I (RG-I), and thus were named pectin O-acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG-I, whereas POAT1,3,7,8 could act on both HG and RG-I. The acetylation of HG and RG-I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG-I. Furthermore, simultaneous RNA interference downregulation of POAT1,3,6,7,8 resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectin O-acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG-I.

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.

Needle guidance with Doppler-tracked polarization-sensitive optical coherence tomography.

Significance: Optical coherence tomography (OCT) can be integrated into needle probes to provide real-time navigational guidance. However, unscanned implementations, which are the simplest to build, often struggle to discriminate the relevant tissues. Aim: We explore the use of polarization-sensitive (PS) methods as a means to enhance signal interpretability within unscanned coherence tomography probes. Approach: Broadband light from a laser centered at 1310 nm was sent through a fiber that was embedded into a needle. The polarization signal from OCT fringes was combined with Doppler-based tracking to create visualizations of the birefringence properties of the tissue. Experiments were performed in (i) well-understood structured tissues (salmon and shrimp) and (ii) ex vivo porcine spine. The porcine experiments were selected to illustrate an epidural guidance use case. Results: In the porcine spine, unscanned and Doppler-tracked PS OCT imaging data successfully identified the skin, subcutaneous tissue, ligament, and epidural spaces during needle insertion. Conclusions: PS imaging within a needle probe improves signal interpretability relative to structural OCT methods and may advance the clinical utility of unscanned OCT needle probes in a variety of applications.

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.

Quantitative polarization-sensitive super-resolution solid immersion microscopy reveals biological tissues' birefringence in the terahertz range.

Terahertz (THz) technology offers a variety of applications in label-free medical diagnosis and therapy, majority of which rely on the effective medium theory that assumes biological tissues to be optically isotropic and homogeneous at the scale posed by the THz wavelengths. Meanwhile, most recent research discovered mesoscale ([Formula: see text]) heterogeneities of tissues; [Formula: see text] is a wavelength. This posed a problem of studying the related scattering and polarization effects of THz-wave-tissue interactions, while there is still a lack of appropriate tools and instruments for such studies. To address this challenge, in this paper, quantitative polarization-sensitive reflection-mode THz solid immersion (SI) microscope is developed, that comprises a silicon hemisphere-based SI lens, metal-wire-grid polarizer and analyzer, a continuous-wave 0.6 THz ([Formula: see text] µm) backward-wave oscillator (BWO), and a Golay detector. It makes possible the study of local polarization-dependent THz response of mesoscale tissue elements with the resolution as high as [Formula: see text]. It is applied to retrieve the refractive index distributions over the freshly-excised rat brain for the two orthogonal linear polarizations of the THz beam, aimed at uncovering the THz birefringence (structural optical anisotropy) of tissues. The most pronounced birefringence is observed for the Corpus callosum, formed by well-oriented and densely-packed axons bridging the cerebral hemispheres. The observed results are verified by the THz pulsed spectroscopy of the porcine brain, which confirms higher refractive index of the Corpus callosum when the THz beam is polarized along axons. Our findings highlight a potential of the quantitative polarization THz microscopy in biophotonics and medical imaging.

Birefringence-derived artifact in optical coherence tomography imaging of the lamina cribrosa in eyes with glaucoma.

We investigated birefringence-derived artifacts that potentially mimic focal defects of the lamina cribrosa (focal LC defects) in optical coherence tomography (OCT) imaging of eyes with glaucoma. This study included 74 eyes of 48 patients with glaucoma. Five horizontal line B-scan images of the optic disc were obtained using commercial swept-source OCT. From a dataset of prototype swept-source polarization-diversity OCT, we calculated the following types of OCT images: polarization-dependent, polarization-dependent attenuation-coefficient, polarization-independent, and polarization-independent attenuation-coefficient. We assessed the commercial OCT images for the presence of birefringence-derived artifacts by comparison with the polarization-diversity OCT images. Commercial OCT showed suggestive findings of focal LC defects in 17 of 74 eyes. Reevaluation using polarization-independent OCT revealed that the focal LC defects in one of 17 eyes (5.9%) were actually birefringence-derived artifacts. This study demonstrated the existence of birefringence-derived artifacts mimicking focal LC defects in commercial OCT imaging and indicated that polarization-diversity OCT is an effective tool to evaluate the presence of these artifacts.

The effect of pupil size on the measurement of corneal birefringence properties: preliminary study.

We used a partial Mueller matrix polarimeter to measure the corneal anisotropic properties at three pupil sizes (dilated, natural, and constricted). The geometrical parameters of first order isochromes were described by quadrilaterals. These parameters are statistically significantly different between the three pupil sizes. The pupillary size changes do not influence the azimuth angle distribution α. The retardation R and birefringence distributions show asymmetry in the nasal-temporal cross-section. There are differences between pupil sizes for both nasal and temporal parts of the cornea for these distributions. Iridial light scattering and diffraction might be the reason for these differences.

Mueller-matrix imaging polarimetry elevated by wavelet decomposition and polarization-singular processing for analysis of specific cancerous tissue pathology.

Significance: Mueller-matrix polarimetry is a powerful method allowing for the visualization of malformations in biological tissues and quantitative evaluation of alterations associated with the progression of various diseases. This approach, in fact, is limited in observation of spatial localization and scale-selective changes in the poly-crystalline compound of tissue samples. Aim: We aimed to improve the Mueller-matrix polarimetry approach by implementing the wavelet decomposition accompanied with the polarization-singular processing for express differential diagnosis of local changes in the poly-crystalline structure of tissue samples with various pathology. Approach: Mueller-matrix maps obtained experimentally in transmitted mode are processed utilizing a combination of a topological singular polarization approach and scale-selective wavelet analysis for quantitative assessment of the adenoma and carcinoma histological sections of the prostate tissues. Results: A relationship between the characteristic values of the Mueller-matrix elements and singular states of linear and circular polarization is established within the framework of the phase anisotropy phenomenological model in terms of linear birefringence. A robust method for expedited (up to ∼15 min) polarimetric-based differential diagnosis of local variations in the poly-crystalline structure of tissue samples containing various pathology abnormalities is introduced. Conclusions: The benign and malignant states of the prostate tissue are identified and assessed quantitatively with a superior accuracy provided by the developed Mueller-matrix polarimetry approach.

Accurate design of a TeO2 noncollinear acousto-optic tunable filter with refractive index correction.

The refractive index is a key factor in the design and analysis of noncollinear acousto-optic tunable filter (AOTF) devices. While previous studies have corrected and analyzed the effects of anisotropic birefringence and the rotatory property, they still rely on paraxial and elliptical approximations, which can introduce non-negligible errors (0.5° or more) into the geometric parameters of TeO2 noncollinear AOTF devices. In this paper, we address these approximations and their effects through refractive index correction. This fundamental theoretical research has significant implications for the design and application of noncollinear AOTF devices.

Polarization-insensitive optical coherence tomography based on partly depolarized light.

Polarization-related image artifacts are frequently observed in optical coherence tomography (OCT) data. As most modern OCT layouts rely on polarized light sources, only the co-polarized component of the light scattered from within a sample can be detected after interference with the reference beam. Cross-polarized sample light does not interfere with the reference beam and thus produces artifacts ranging from a reduction to the full absence of OCT signals. Here we present a simple yet effective technique to prevent polarization artifacts. By partly depolarizing the light source at the interferometer entrance, we achieve OCT signals regardless of the sample polarization state. We demonstrate the performance of our approach in a defined retarder as well as in birefringent dura mater tissue. This simple and cost-effective technique can be applied to obviate cross-polarization artifacts in virtually any OCT layout.

Birefringence analysis of collagen supraorganization in cat corneas with tropical keratopathy.

OBJECTIVE: To evaluate the birefringent properties of the cornea and examine the supraorganizational aspects of collagen fibers in cats with tropical keratopathy. PROCEDURE: In this study, 10-micrometer-thick sections of corneal tissue from cats with tropical keratopathy were examined, both in the opaque and transparent areas of the anterior stroma. Control samples were obtained from healthy cat corneas. Polarized light microscopy was employed to evaluate the birefringent properties using two distinct methods. The first method involved measuring the optical retardation associated with corneal birefringence, while the second method assessed the alignment/waviness of the birefringent collagen fibers. Differences were significant when p < .05. RESULTS: Tropical keratopathy resulted in a significant rise (p < .05) in optical retardation in both opaque and transparent regions of the cat cornea. In the anterior stroma, both the opaque zones and transparent tissue exhibited a higher degree of collagen fiber packing than the control corneas. However, no significant differences (p > .05) in alignment were observed between the transparent tissue of the diseased cornea and the healthy corneas. CONCLUSION: Supraorganizational changes in collagen fiber packing are not restricted to lesion zones in cat corneas affected by tropical keratopathy. Such alterations also occur in the corneal tissue of the anterior stroma adjoining the lesions. Therefore, it is plausible that the transparent tissue of the anterior stroma in corneas affected by the disease may have functional abnormalities, despite its macroscopic healthy appearance. Additional investigations are required to clarify the implications of these potential defects and their conceivable contribution to tropical keratopathy.

Posterior scleral birefringence measured by triple-input polarization-sensitive imaging as a biomarker of myopia progression.

In myopic eyes, pathological remodelling of collagen in the posterior sclera has mostly been observed ex vivo. Here we report the development of triple-input polarization-sensitive optical coherence tomography (OCT) for measuring posterior scleral birefringence. In guinea pigs and humans, the technique offers superior imaging sensitivities and accuracies than dual-input polarization-sensitive OCT. In 8-week-long studies with young guinea pigs, scleral birefringence was positively correlated with spherical equivalent refractive errors and predicted the onset of myopia. In a cross-sectional study involving adult individuals, scleral birefringence was associated with myopia status and negatively correlated with refractive errors. Triple-input polarization-sensitive OCT may help establish posterior scleral birefringence as a non-invasive biomarker for assessing the progression of myopia.

Mueller matrix imaging of pathological slides with plastic coverslips.

Mueller matrix microscopy is capable of polarization characterization of pathological samples and polarization imaging based digital pathology. In recent years, hospitals are replacing glass coverslips with plastic coverslips for automatic preparations of dry and clean pathological slides with less slide-sticking and air bubbles. However, plastic coverslips are usually birefringent and introduce polarization artifacts in Mueller matrix imaging. In this study, a spatial frequency based calibration method (SFCM) is used to remove such polarization artifacts. The polarization information of the plastic coverslips and the pathological tissues are separated by the spatial frequency analysis, then the Mueller matrix images of pathological tissues are restored by matrix inversions. By cutting two adjacent lung cancer tissue slides, we prepare paired samples of very similar pathological structures but one with a glass coverslip and the other with a plastic coverslip. Comparisons between Mueller matrix images of the paired samples show that SFCM can effectively remove the artifacts due to plastic coverslip.

Development of an intelligent full-field polarization sensitive optical coherence tomography for breast cancer classification.

Biopsy is the gold standard for cancer detection, however the surge in breast cancer cases has made manual haematoxylin and eosin stained histopathological image examination difficult. Automatic cancer diagnosis is vital for a healthy life. It allows fast diagnosis without specific skills. This research proposes an intelligent full-field polarization-sensitive optical coherence tomography (FF-PS-OCT) system for ex-vivo breast classification using ensemble model corroborated by technique for order preference by similarity to ideal solution (TOPSIS). 220 samples image were scanned using the FF-PS-OCT to extract the phase information. The multilevel ensemble classifier has 94.8% precision, 92.5% recall, 93.7% F-score and 82.3% Mathews correlation coefficient on the testing dataset. The developed ensemble model corroborated by TOPSIS, outperforms the single model in terms of performance metrics. The initial results indicate that the rapid, non-contact and label-free FF-PS-OCT imaging modality using birefringent information is beneficial for making interventional decisions by clinicians.

Dual-slit confocal light-sheet microscopy using birefringent crystals.

We present a method to use a birefringent crystal for generating two illumination beams in a digital scanned laser light-sheet microscopy (DSLM) system. Upon this, a conventional confocal DSLM can be easily upgraded to a dual-slit confocal DSLM with two-fold imaging speed. We have implemented this method to our bidirectional DSLM system, locating two identical calcite crystals on both illumination paths from both sides of the sample. The neurons of in vivo larval zebrafish have been fast imaged with sterling image quality, especially ~2.5 times higher contrast, compared to the conventional DSLM.