Transparent Specimens Research Articles

Dielectric Metasurfaces for Broadband Phase-Contrast Relief-Like Imaging.

The visualization of transparent specimens in traditional light microscopy is impeded by insufficient intrinsic contrast, prompting the development of advanced contrast-enhancement methodologies to transmute minute phase discrepancies into detectable amplitude alterations. While existing methods excel in either phase-contrast imaging (contrast-enhanced image of whole objects) or relief-like imaging (deceptive three-dimensional images), it would be of great significance to seamlessly integrate both capabilities in the same device. Here, we propose a novel metasurface-assisted half-side phase-contrast technique capable of simultaneous phase-contrast and relief-like imaging across the visible spectrum, which is realized by introducing a ±π/2 phase shift to a half-side diffracted wave emitted by the objects. Our method showcases successful application to diverse specimens, including a transparent silica disk and a frog egg cell. Our work substantiates high-quality microscopic imaging of various transparent specimens, which has profound implications in cellular biology, materials science, and medical diagnostics.

Compact common-path polarization holography for measurement of the Jones matrix of polarization-sensitive materials

A compact common-path off-axis digital holographic imaging method is proposed utilizing polarization-angular-multiplexing for Jones matrix measurement. Our method employs a common-path off-axis configuration to capture multiplexed off-axis interferograms generated by orthogonally polarized object beams and a reference beam on a monochrome CCD camera. The modulation of the fringe direction is achieved by two homemade retro-reflector mirrors, allowing for the retrieval of the Jones matrix distribution of transparent specimens through a matrix-division algorithm. The stable common-path design and the expansive camera field of view facilitate the extraction of spatially resolved Jones matrix parameters. The feasibility of this method was validated through experiments involving standard objects and polarization-sensitive materials, conducted at both general and microscopic scales. Our system completed polarization imaging of cancerous tissues, unequivocally demonstrating its extraordinary potential in medical pathology diagnostics.

Beyond comparison: Brillouin microscopy and AFM-based indentation reveal divergent insights into the mechanical profile of the murine retina

Mechanical tissue properties increasingly serve as pivotal phenotypic characteristics that are subject to change during development or pathological progression. The quantification of such material properties often relies on physical contact between a load-applying probe and an exposed sample surface. For most tissues, meeting these requirements entails an invasive preparation, which poses the risk of yielding mechanical properties that do not portray the physiological state of a tissue within a functioning organism. Brillouin microscopy has emerged as a non-invasive, optical technique that enables the assessment of mechanical cell and tissue properties with high spatio-temporal resolution. In optically transparent specimens, it does not require animal sacrifice, tissue dissection or sectioning. However, the extent to which results obtained from Brillouin microscopy allow to infer conclusions about potential results obtained with a contact-based technique, and vice versa, is unclear. Sources for discrepancies include the varying characteristic temporal and spatial scales, the directionality of measurement, environmental factors, and mechanical moduli probed. In this work, we addressed those aspects by quantifying the mechanical properties of acutely dissected murine retinae using Brillouin microscopy and atomic force microscopy (AFM)-based indentation measurements. Our results show a distinct mechanical profile of the retinal layers with respect to the Brillouin frequency shift, the Brillouin linewidth and the apparent Young’s modulus. Contrary to previous reports, our findings do not support a simple correlative relationship between Brillouin frequency shift and apparent Young’s modulus. Additionally, the divergent sensitivities of Brillouin microscopy and AFM-indentation measurements to structural features, as visualized by transmission electron microscopy, to cross-linking or changes post mortem underscore the dangers of assuming interchangeability between the two methods. In conclusion, our study advocates for viewing Brillouin microscopy and AFM-based indentation measurements as complementary tools, discouraging direct comparisons a priori and suggesting their combined use for a more comprehensive understanding of tissue mechanical properties.

Concept for improving the form measurement results of aspheres and freeform surfaces in a tilted-wave interferometer

Abstract. Accurate and flexible form measurements for aspherical and freeform surfaces are in high demand, and non-null-test interferometric methods such as tilted-wave interferometry have gained attention as a promising response to this need. Interferometric methods, however, display ambiguities between the measurement of certain form errors and the misalignment of the measured specimen. Therefore, improved knowledge of the absolute measurement position of the specimen in relation to the interferometer setup may improve the form measurement result. In this work, we propose a concept that uses a white light interferometer to measure the absolute distance between a transparent specimen's surface and the interferometer's objective and present preparatory data to qualify the white light interferometer for the improvement of tilted-wave interferometer measurements.

Speckle-enabled in vivo demixing of neural activity in the mouse brain.

Functional imaging of neuronal activity in awake animals, using a combination of fluorescent reporters of neuronal activity and various types of microscopy modalities, has become an indispensable tool in neuroscience. While various imaging modalities based on one-photon (1P) excitation and parallel (camera-based) acquisition have been successfully used for imaging more transparent samples, when imaging mammalian brain tissue, due to their scattering properties, two-photon (2P) microscopy systems are necessary. In 2P microscopy, the longer excitation wavelengths reduce the amount of scattering while the diffraction-limited 3D localization of excitation largely eliminates out-of-focus fluorescence. However, this comes at the cost of time-consuming serial scanning of the excitation spot and more complex and expensive instrumentation. Thus, functional 1P imaging modalities that can be used beyond the most transparent specimen are highly desirable. Here, we transform light scattering from an obstacle into a tool. We use speckles with their unique patterns and contrast, formed when fluorescence from individual neurons propagates through rodent cortical tissue, to encode neuronal activity. Spatiotemporal demixing of these patterns then enables functional recording of neuronal activity from a group of discriminable sources. For the first time, we provide an experimental, in vivo characterization of speckle generation, speckle imaging and speckle-assisted demixing of neuronal activity signals in the scattering mammalian brain tissue. We found that despite an initial fast speckle decorrelation, substantial correlation was maintained over minute-long timescales that contributed to our ability to demix temporal activity traces in the mouse brain in vivo. Informed by in vivo quantifications of speckle patterns from single and multiple neurons excited using 2P scanning excitation, we recorded and demixed activity from several sources excited using 1P oblique illumination. In our proof-of-principle experiments, we demonstrate in vivo speckle-assisted demixing of functional signals from groups of sources in a depth range of 220-320 µm in mouse cortex, limited by available speckle contrast. Our results serve as a basis for designing an in vivo functional speckle imaging modality and for maximizing the key resource in any such modality, the speckle contrast. We anticipate that our results will provide critical quantitative guidance to the community for designing techniques that overcome light scattering as a fundamental limitation in bioimaging.

Proportioning optimization of transparent rock-like specimens with different fracture structures

Clarifying the principles of proportioning optimization for brittle transparent rock-like specimens with differential fracture structures is crucial for the visualization study of the internal fracture and seepage evolution mechanisms in rock masses. This study, utilizing orthogonal experimental methods, uncovers the influence mechanisms, extents, and patterns by which the ratios of resin, hardener, and accelerator, along with the freezing duration, impact the mechanical characteristics of transparent rock-like specimens. Notably, it was observed that as the accelerator ratio and freezing time are increased, there’s a general decline in the uniaxial compressive strength, tensile strength, and elastic modulus of the specimens. In contrast, an increase in the hardener ratio initially leads to an enhancement in these mechanical properties, followed by a subsequent decrease. Under uniaxial compressive loading, the specimens exhibit four typical modes of failure: bursting failure, splitting failure, single inclined plane failure, and bulging failure. As the hardener and accelerator ratios increase, the mode of failure gradually shifts from bulging to bursting, with freezing time having a minor overall impact on the evolution of failure modes. The study proposes a method for inducing random three-dimensional closed fractures within the specimens and further clarifies the principles for optimizing the proportions of specimens with different fracture structures, such as intact, embedded regular, and random three-dimensional fractures. This research facilitates the in-depth application of transparent rock-like materials in various scenarios and provides theoretical guidance and technical support for visualizing the evolution of fracture and seepage characteristics within the fractured rock mass.

Electrically-switched differential microscopy based on computing liquid-crystal platforms

Abstract Detection of transparent phase specimens especially biological cells with desired contrasts is of great importance in visual display and medical diagnosis. Due to the pure-phase nature, conventional detection approaches may damage samples or require complex operations. Computing liquid crystal (LC) is a thin and flat optical element with excellent capability in optical field modulation, which gives a feasible way to this issue from the perspective of analog optical computing. We here propose and experimentally implement an electrically switched two-dimensional (2D) differential microscopy based on computing LC platforms. The Pancharatnam–Berry phase LC polarization grating induces light’s spin separation to promote the 2D differential operation. Using the electrically tunable LC plate as the system phase retardance provider, the detecting mode can be flexibly switched from bright-field images to edge-enhanced images with desired contrasts. Remarkably, owing to the wavelength-independent feature closely related to the geometric phases, our proposed scheme is demonstrated to be applicable to the multi-wavelength microscopy imaging. These results open avenues to form real-time all-optical image processing and may facilitate multifunctional differential microscopy.

Super resolution label-free dark-field microscopy by deep learning.

Dark-field microscopy (DFM) is a powerful label-free and high-contrast imaging technique due to its ability to reveal features of transparent specimens with inhomogeneities. However, owing to the Abbe's diffraction limit, fine structures at sub-wavelength scale are difficult to resolve. In this work, we report a single image super resolution DFM scheme using a convolutional neural network (CNN). A U-net based CNN is trained with a dataset which is numerically simulated based on the forward physical model of the DFM. The forward physical model described by the parameters of the imaging setup connects the object ground truths and dark field images. With the trained network, we demonstrate super resolution dark field imaging of various test samples with twice resolution improvement. Our technique illustrates a promising deep learning approach to double the resolution of DFM without any hardware modification.

Anatomical study of the architecture of the cutaneous branch-chained blood vessels in the medial lower leg in adult

Objective: To investigate the architecture of the cutaneous branch-chained blood vessels in the medial lower leg and provide the anatomical basis for design and clinical application of the cutaneous branch-chained flap from this region. Methods: The experimental research method was used. From March to May 2023, the anatomical study was conducted on the 5 voluntarily donated fresh adult (aged 50 to 70 years, all male) cadaveric specimens from Hangzhou Normal University School of Basic Medical Sciences. The fine anatomy under microscope was performed on each lower leg specimens of 5 corpses (1 lower leg specimen was conducted with digital radiography (DR) scan before fine anatomy), to observe, measure, and record the course of posterior tibial artery, quantity of perforator, the distance between the perforating point of each perforator and the medial condyle of tibia, the external diameter of posterior tibial artery perforator, the length of perforator pedicle, the horizontal distance between the posterior tibial artery perforator and the saphenous nerve, and the course of each perforator within superficial fascia after crossing the deep fascia and the distribution of the cutaneous branch-chains. The DR scan under the perfusion of barium sulfate was conducted in one lower leg specimen to observe the distribution of cutaneous branch-chained vascular network (hereinafter referred to as vascular chain) between perforators. Transparent skin specimen was made from one leg specimen after anatomy to observe the distribution of perforators and vascular chains between perforators. Results: In 5 lower leg specimens, the upper part of posterior tibial artery was located deep in soleus muscle, and the lower part was located between the medial edge of gastrocnemius muscle and flexor digitorum longus muscle. A total of 28 posterior tibial artery perforators were identified, with an average of 5.6 branches in each lower leg. The distance between the perforating point of perforator and the medial condyle of tibia ranged from 6.5 to 36.0 cm, mainly distributed at 22.0 (15.1, 28.1) cm from the medial condyle of tibia, in zones 3 to 6. The external diameters of perforators of posterior tibial arteries were 0.7-1.1 mm. The length of perforator pedicle was 1.0-4.5 cm, and the horizontal distance between the posterior tibial artery perforator and the saphenous nerve was 0.5-3.0 cm. The fine anatomy under microscope showed that the posterior tibial artery perforators had long upward and downward branches after crossing the deep fascia, and the ascending branches and descending branches were anastomosed longitudinally to form the nutrient cutaneous branch-chain in the medial lower leg. DR scan and transparent skin specimen both showed that longitudinal vascular chain was formed between the posterior tibial artery perforators, the transparent skin specimen also showed that longitudinal blood vessel chains included the direct connecting vessels in the adipose layer and the indirect connecting vessels in the subdermal layer. Conclusions: The cutaneous branch-chained vessels in the medial lower leg are constructed by posterior tibial artery perforators, direct connecting vessels, indirect connecting vessels, and traffic branches. The cutaneous branch-chained flap is reliable in terms of vascular anatomy, and can carry the saphenous nerve for partial restoration of its sensation, thus it is an ideal flap.

Dielectric Metasurface Enabled Compact, Single-Shot Digital Holography for Quantitative Phase Imaging.

Quantitative phase imaging (QPI) enables nondestructive, real-time, label-free imaging of transparent specimens and can reveal information about their fundamental properties such as cell size and morphology, mass density, particle dynamics, and cellular fluctuations. Development of high-performance and low-cost quantitative phase imaging systems is thus required in many fields, including on-site biomedical imaging and industrial inspection. Here, we propose an ultracompact, highly stable interferometer based on a single-layer dielectric metasurface for common path off-axis digital holography and experimentally demonstrate quantitative phase imaging. The interferometric imaging system leveraging an ultrathin multifunctional metasurface captures image plane holograms in a single shot and provides quantitative phase information on the test samples for extraction of its physical properties. With the benefits of planar engineering and high integrability, the proposed metasurface-based method establishes a stable miniaturized QPI system for reliable and cost-effective point-of-care devices, live cell imaging, 3D topography, and edge detection for optical computing.

A double-sided surface scanning platform for sapphire substrate quality assessment

With excellent mechanical and optical properties, sapphire substrate becomes a very important industrial material especially for LED manufacturing. Its quality control requires two tasks, double-sided surface profiling and defect detection. In this paper, a 3D surface scanning system is proposed as a platform for sapphire substrate inspection. During the scanning process, the height information of both upper and lower surfaces is measured by a chromatic confocal probe. For lateral positioning feedback, an in-house developed sensor, namely, image grating, is introduced in this paper. For these inspection processes, there are three challenges: 1) the motion error in the vertical direction, 2) simultaneous measurement of both upper and lower surfaces, and 3) lateral positioning error in a large scanning area. To address these challenges, three key technologies were developed: 1) real-time motion error compensation based on a fringe interferometer, 2) a double-sided measurement for transparent specimen without the pre-knowledge of its refractive index, and 3) planar image grating pair for accurate 2D displacement feedback. The experimental results showed that the proposed measurement system was able to achieve 50 nm accuracy of the standard deviation when measuring the step height of gauge blocks. When the system measured 4-inch double-sided polished sapphire substrate, the measurement repeatability of TTV (Total Thickness Variation) was within 60 nm; and that of warpage was within 10 nm. As a side product, refractive index can also be measured using the proposed system, and experimental results showed respectable consistency.

Multispectral Quantitative Phase Imaging Using a Diffractive Optical Network

As a label‐free imaging technique, quantitative phase imaging (QPI) provides optical path length information of transparent specimens for various applications in biology, materials science, and engineering. Multispectral QPI measures quantitative phase information across multiple spectral bands, permitting the examination of wavelength‐specific phase and dispersion characteristics of samples. Herein, the design of a diffractive processor is presented that can all‐optically perform multispectral quantitative phase imaging of transparent phase‐only objects within a snapshot. The design utilizes spatially engineered diffractive layers, optimized through deep learning, to encode the phase profile of the input object at a predetermined set of wavelengths into spatial intensity variations at the output plane, allowing multispectral QPI using a monochrome focal plane array. Through numerical simulations, diffractive multispectral processors are demonstrated to simultaneously perform quantitative phase imaging at 9 and 16 target spectral bands in the visible spectrum. The generalization of these diffractive processor designs is validated through numerical tests on unseen objects, including thin Pap smear images. Due to its all‐optical processing capability using passive dielectric diffractive materials, this diffractive multispectral QPI processor offers a compact and power‐efficient solution for high‐throughput quantitative phase microscopy and spectroscopy.

Deep learning-based quantitative phase microscopy

Quantitative phase microscopy (QPM) is a powerful tool for label-free and noninvasive imaging of transparent specimens. In this paper, we propose a novel QPM approach that utilizes deep learning to reconstruct accurately the phase image of transparent specimens from a defocus bright-field image. A U-net based model is used to learn the mapping relation from the defocus intensity image to the phase distribution of a sample. Both the off-axis hologram and defocused bright-field image are recorded in pair for thousands of virtual samples generated by using a spatial light modulator. After the network is trained with the above data set, the network can fast and accurately reconstruct the phase information through a defocus bright-field intensity image. We envisage that this method will be widely applied in life science and industrial detection.

Quantification of geogrid lateral restraint using transparent sand and deep learning-based image segmentation

An experimental technique is presented to quantify the lateral restraint provided by a geogrid embedded in granular soil at the particle level. Repeated load triaxial tests were done on transparent sand specimens stiffened with geosynthetic inclusions simulating geogrids. Images of laser illuminated planes through the specimens were segmented into particles using a deep learning-based segmentation algorithm. The particle outlines were characterized in terms of Fourier shape descriptors and tracked across sequentially captured images. The accuracy of the particle displacement measurements was validated against Digital Image Correlation (DIC) measurements. In addition, the method's resolution and repeatability is presented. Based on the measured particle displacements and rotations, a state boundary line between probable and improbable particle motions was identified for each test. The size of the zone of probable motions was used to quantify the lateral restraint provided by the inclusions. Overall, the tests results revealed that the geosynthetic inclusions restricted both particle displacements and rotations. However, the particle displacements were found to be restrained more significantly than the rotations. Finally, a unique relationship was found between the magnitude of the permanent strains of the specimens and the area of the zone of probable motions.

An optical method to monitor transparent contact interfaces during high frequency shear vibration cycles

Contacting interfaces provide frictional damping in jointed structures subjected to high dynamic loads. Predicting this frictional damping during vibration cycles is highly important since it strongly affects the dynamic response of the assembly and hence the lifetime of parts. Since frictional damping is heavily influenced by the contact condition at the interface, the most direct and insightful approach is thereby to actively monitor the contact interface. Although several methods have already been proposed to monitor the contact interfaces quasi-statically or in pre-sliding, such as digital image correlation, X-Ray and ultrasound, only limited data is available of the frictional interface behaviour during high frequency vibration.To provide a better insight into the contact interface behaviour during high frequency cyclic motion, an optical method is here developed based on transparent friction specimens and total internal reflection, and applied to an existing friction test rig. The resulting measurements across the whole interface show a large variation in the real area of contact during each vibration cycle, which could be linked to the kinematics of the contact interface. This large variation is observed for the first time in high frequency oscillating contacts and is attributed to ageing effect and fracture of asperities. These two effects dominate the contact mechanism at different sliding velocities and induce variations in the real area of contact during each vibration cycle. These results suggest that the mechanisms behind high frequency contact behaviour are more complex than what commonly assumed in dynamics simulations.

Reflective Phase-Contrast for High-Contrast Imaging of van der Waals Heterostructure

Optical microscopy plays a critical role in the fabrication of two-dimensional (2D) van der Waals heterostructures. An outstanding challenge in conventional microscopy is to visualize transparent 2D layers as well as embedded monolayers in a stacked heterostructure with high optical contrast. Phase-contrast microscopy, first developed by Frits Zernike in the 1930s, leverages the interference effect between specimen scattered light and background light to increase the contrast of transparent specimens. Such phase-contrast microscopy, always in a transmission configuration, revolutionized the study of transparent cellular structures in biology. Here, we develop a versatile reflective phase-contrast microscopy for imaging 2D heterostructures. We employ two spatial light modulators to flexibly control the intensity and phase of the illumination and the reflected light. This reflective phase-contrast microscopy achieves unprecedented high contrast for imaging a transparent 2D monolayer. It also enables direct observation of 2D monolayers embedded inside a thick heterostructure that are "invisible" in conventional microscopy.

Effect of shear-induced contact area and aperture variations on nonlinear flow behaviors in fractal rock fractures

This study experimentally analyzes the nonlinear flow characteristics and channelization of fluid through rough-walled fractures during the shear process using a shear-flow-visualization apparatus. A series of fluid flow and visualization tests is performed on four transparent fracture specimens with various shear displacements of 1 mm, 3 mm, 5 mm, 7 mm and 10 mm under a normal stress of 0.5 MPa. Four granite fractures with different roughnesses are selected and quantified using variogram fractal dimensions. The obtained results show that the critical Reynolds number tends to increase with increasing shear displacement but decrease with increasing roughness of fracture surface. The flow paths are more tortuous at the beginning of shear because of the wide distribution of small contact spots. As the shear displacement continues to increase, preferential flow paths are more distinctly observed due to the decrease in the number of contact spots caused by shear dilation; yet the area of single contacts increases. Based on the experimental results, an empirical mathematical equation is proposed to quantify the critical Reynolds number using the contact area ratio and fractal dimension. • A visualization method is developed to determine the channelization of sheared fracture. • A model for predicting the Re c is proposed.

Method for visualizing the shear process of rock joints using 3D laser scanning and 3D printing techniques

This study presents a visualized approach for tracking joint surface morphology. Three-dimensional laser scanning (3DLS) and 3D printing (3DP) techniques are adopted to record progressive failure during rock joint shearing. The 3DP resin is used to create transparent specimens to reproduce the surface morphology of a natural joint precisely. The freezing method is employed to enhance the mechanical properties of the 3DP specimens to reproduce the properties of hard rock more accurately. A video camera containing a charge-coupled device (CCD) camera is utilized to record the evolution of damaged area of joint surface during the direct shear test. The optimal shooting distance and shooting angle are recommended to be 800 mm and 40°, respectively. The images captured by the CCD camera are corrected to quantitatively describe the damaged area on the joint surface. Verification indicates that this method can accurately describe the total sheared areas at different shear stages. These findings may contribute to elucidating the shear behavior of rock joints.

Design and Research of Chromatic Confocal System for Parallel Non-Coaxial Illumination Based on Optical Fiber Bundle.

Conventional chromatic confocal systems are mostly single-point coaxial illumination systems with a low signal-to-noise ratio, light energy utility and measurement efficiency. To overcome the above shortcomings, we propose a parallel non-coaxial-illumination chromatic-confocal-measurement system based on an optical fiber bundle. Based on the existing single-point non-coaxial-illumination system, the optical fiber bundle is used as the optical beam splitter to achieve parallel measurements. Thus, the system can yield measurements through line scanning, which greatly improves measurement efficiency. To verify the measurement performance of the system, based on the calibration experiment, the system realizes the measurement of the height of the step, the thickness of the transparent specimen and the reconstruction of the three-dimensional topography of the surface of the step and coin. The experimental results show that the measuring range of the system is 200 μm. The measurement accurcy can reach micron level, and the system can realize a good three-dimensional topography reconstruction effect.

Iodonium Borate Initiators for Cationic Photopolymerization and Their Application in Radical-Induced Cationic Frontal Polymerization

Iodonium antimonates and borates are well established initiators in the field of cationic photopolymerization and preferred substances for radical-induced cationic frontal polymerization (RICFP). Extensive research in the fields of ionic liquids and lithium ion battery electrolytes made many superacids based on the borate anions available. Cyanide-ligated borane B2 has lower molecular weight compared to state-of-the-art borates and is less expensive to synthesize from commercial starting materials. By comparing reactivity in the epoxy-based monomer bisphenol-A-diglycidylether, B2 outperforms all commercial systems in a broad range of temperatures with excellent epoxy group conversions of up to 95%. Besides its good performance as a cationic initiator, B2 is also capable of thermally and photochemically induced RICFP. Exceptional are transparent B2 specimens in contrast to the tested commercial systems, which are black. High reactivity, good frontal parameters, easy synthetic access, and sufficient pot life of formulations make cyanide-ligated iodonium borates a compelling alternative to current state-of-the-art initiators.