Molecular mechanisms of cardiac actomyosin transforming from rigor state to post-rigor state.

Sudden cardiac death contributed to half of all deaths from cardiovascular diseases. The mechanism of the kinetic cycle of cardiac myosin is crucial for heart protection and drug development. The state change in the myosin kinetic cycle from the rigor state to the post-rigor state is fundamental to explain binding and dissociation. Here, we used ß-cardiac myosin in the rigor and post-rigor states to model the actomyosin complexes. Molecular dynamics simulations, electrostatic analysis, and energetic analysis of actomyosin complexes were performed in this work. The results showed that there are fewer interactions and lower electrostatic binding strength in the post-rigor state than in the rigor state. In the post-rigor state, there were higher free binding energy, fewer salt bridges, and fewer hydrogen bonds. The results showed a lower binding affinity in the post-rigor state than in the rigor state. The decrease in the binding affinity provided important conditions for dissociation of the myosin from the actin filament. Although previous studies focused mostly on the binding process, this study provides evidence of dissociation, which is even more important in the myosin kinetic cycle. This research on the mechanism of myosin kinetic cycles provides a novel direction for future genetic disease studies.

High-efficiency 3D reconstruction with a uniaxial MEMS-based fringe projection profilometry.

Micro-Electro-Mechanical System (MEMS) scanning is increasingly popular in 3D surface measurement with the merits of the compact structure and high frame-rate. In this paper, we achieve real-time fringe structured 3D reconstruction by using a uniaxial MEMS-based projector. To overcome the limitations on uniaxial MEMS-based projector of lensless structure and unidirectional fringe projection, a novel isophase plane model is proposed, in which the laser line from MEMS-based projector is regarded as an isophase plane. Our model directly establishes the mapping relationship between phase and spatial 3D coordinates through the intersection point of camera back-projection light ray and isophase plane. Furthermore, a flexible calibration strategy to obtain 3D mapping coefficients is introduced with a specially designed planar target. Experiments demonstrated that our method can achieve high-accuracy and real-time 3D reconstruction.

Study of the Expression Transition of Cardiac Myosin Using Polarization-Dependent SHG Microscopy.

Detection of the transition between the two myosin isoforms α- and ß-myosin in living cardiomyocytes is essential for understanding cardiac physiology and pathology. In this study, the differences in symmetry of polarization spectra obtained from α- and ß-myosin in various mammalian ventricles and propylthiouracil-treated rats are explored through polarization-dependent second harmonic generation microscopy. Here, we report for the, to our knowledge, first time that α- and ß-myosin, as protein crystals, possess different symmetries: the former has C6 symmetry, and the latter has C3v. A single-sarcomere line scan further demonstrated that the differences in polarization-spectrum symmetry between α- and ß-myosin came from their head regions: the head and neck domains of α- and ß-myosin account for the differences in symmetry. In addition, the dynamic transition of the polarization spectrum from C6 to C3v line profile was observed in a cell culture in which norepinephrine induced an α- to ß-myosin transition.

Ray calibration and phase mapping for structured-light-field 3D reconstruction.

In previous work, we presented a structured light field (SLF) method combining light field imaging with structured illumination to perform multi-view depth measurement. However, the previous work just accomplishes depth rather than 3D reconstruction. In this paper, we propose a novel active method involving ray calibration and phase mapping, to achieve SLF 3D reconstruction. We performed the ray calibration for the first time to determine each light field ray with metric spatio-angular parameters, making the SLF realize multi-view 3D reconstruction. Based on the ray parametric equation, we further derived the phase mapping in the SLF that spatial coordinates can be directly mapped from phase. A flexible calibration strategy was correspondently designed to determine mapping coefficients for each light field ray, achieving high-efficiency SLF 3D reconstruction. Experimental results demonstrated that the proposed method was suitable for high-efficiency multi-view 3D reconstruction in the SLF.

Light-field-based absolute phase unwrapping.

Ambiguity caused by a wrapped phase is an intrinsic problem in fringe projection-based 3D shape measurement. Among traditional methods for avoiding phase ambiguity, spatial phase unwrapping is sensitive to sensor noise and depth discontinuity, and temporal phase unwrapping requires additional encoding information that leads to an increase of image sequence acquisition time or a reduction of fringe contrast. Here, to the best of our knowledge, we report a novel method of absolute phase unwrapping based on light field imaging. In a recorded light field under structured illumination, i.e., a structured light field, a wrapped phase-encoded field can be retrieved and resampled in diverse image planes associated with several possible fringe orders in a measurement volume. Then, by leveraging phase consistency constraint in the resampled wrapped phase-encoded field, correct fringe orders can be determined to unwrap the wrapped phase without any additional encoding information. Experimental results demonstrated that the proposed method was suitable for accurate and robust absolute phase unwrapping.

Universal phase-depth mapping in a structured light field.

Technologies and devices for light field imaging have recently been developed for both industrial applications and scientific research to achieve excellent imaging properties. In our previous work, we combined light field imaging with structured illumination to propose a structured light field method in which multidirectional depth estimation can be performed for high-quality 3D imaging. However, the projection axis was implicitly assumed to be perpendicular to the reference plane, which is hard to meet in practice. In this paper, we derive a universal phase-depth mapping in a structured light field by relaxing this implicit condition. Both nonlinear and linear models were proposed based on this universal relationship. To test the model's practical performance, we simulated experiments by adding errors to the real measured values to evaluate the deviation in depth estimation. By comparing the root-mean-square distributions of the depth deviations with respect to the depth positions, we demonstrated that the nonlinear model was precise and consistent in a wide range of depth, and we employed this model to realize high-quality multidirectional scene reconstruction.

Phase-3D mapping method developed from back-projection stereovision model for fringe projection profilometry.

Two major methods for 3D reconstruction in fringe projection profilometry, phase-height mapping and stereovision, have their respective problems: the former has low-flexibility in practical application due to system restrictions and the latter requires time-consuming homogenous points searching. Given these limitations, we propose a phase-3D mapping method developed from back-projection stereovision model to achieve flexible and high-efficient 3D reconstruction for fringe projection profilometry. We showed that all dimensional coordinates (X, Y, and Z), but not just the height coordinate (Z), of a measured point can be mapped from phase through corresponding rational functions directly and independently. To determine the phase-3D mapping coefficients, we designed a flexible two-step calibration strategy. The first step, ray reprojection calibration, is to determine the stereovision system parameters; the second step, sampling-mapping calibration, is to fit the mapping coefficients using the calibrated stereovision system parameters. Experimental results demonstrated that the proposed method was suitable for flexible and high-efficient 3D reconstruction that eliminates practical restrictions and dispenses with the time-consuming homogenous point searching.

Structured light field 3D imaging.

In this paper, we propose a method by means of light field imaging under structured illumination to deal with high dynamic range 3D imaging. Fringe patterns are projected onto a scene and modulated by the scene depth then a structured light field is detected using light field recording devices. The structured light field contains information about ray direction and phase-encoded depth, via which the scene depth can be estimated from different directions. The multidirectional depth estimation can achieve high dynamic 3D imaging effectively. We analyzed and derived the phase-depth mapping in the structured light field and then proposed a flexible ray-based calibration approach to determine the independent mapping coefficients for each ray. Experimental results demonstrated the validity of the proposed method to perform high-quality 3D imaging for highly and lowly reflective surfaces.

Interactive relationship between basement-membrane development and sarcomerogenesis in single cardiomyocytes.

The cardiac basement membrane (BM), the highly organized layer of the extracellular matrix (ECM) on the external side of the sarcolemma, is mainly composed of laminin and collagen IV, which assemble a dense, well-organized network to surround the surface of each adult cardiomyocyte. The development of the cardiac BM plays a key role in organogenesis of the myocardium through interactions between sarcomeres and integrins. Because of the complicated structure of cardiac muscle fibers and lack of a proper investigation method, the detailed interactions among BM development, sarcomeric growth, and integrin expression remain unclear. In this study, freshly isolated 3-day neonatal cardiomyocytes (CMs) were cultured on aligned collagen, which mimics the in vivo ECM structure and induces neonatal CMs to grow into rod-like shapes. Then double fluorescence-immunostained laminin and α-actinin or integrin ß1 on neonatal CMs cultured 4-72 h were imaged using a confocal microscope, and the spatial relationship between laminin deposition and α-actinin expression was evaluated by colocalization analysis. At 4h, laminin was deposited around Z-bodies (dot-shaped α-actinin) and integrins; from 18-to-72 h, its gradual colocalization with Z-lines (line-shaped α-actinin) and integrins increased Pearson׳s coefficient; this indicates that development of the BM network from the neonatal stage to adulthood is closely related to sarcomeric formation via integrin-mediated interactions.

Microelectrode Array-evaluation of Neurotoxic Effects of Magnesium as an Implantable Biomaterial.

Magnesium (Mg)-based biomaterials have shown great potential in clinical applications. However, the cytotoxic effects of excessive Mg2+ and the corrosion products from Mg-based biomaterials, particularly their effects on neurons, have been little studied. Although viability tests are most commonly used, a functional evaluation is critically needed. Here, both methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to test the effect of Mg2+ and Mg-extract solution on neuronal viability. Microelectrode arrays (MEAs), which provide long-term, real-time recording of extracellular electrophysiological signals of in vitro neuronal networks, were used to test for toxic effects. The minimum effective concentrations (ECmin) of Mg2+ from the MTT and LDH assays were 3 mmol/L and 100 mmol/L, respectively, while the ECmin obtained from the MEA assay was 0.1 mmol/L. MEA data revealed significant loss of neuronal network activity when the culture was exposed to 25% Mg-extract solution, a concentration that did not affect neuronal viability. For evaluating the biocompatibility of Mg-based biomaterials with neurons, MEA electrophysiological testing is a more precise method than basic cell-viability testing.

Fringe projection 3D microscopy with the general imaging model.

Three-dimensional (3D) imaging and metrology of microstructures is a critical task for the design, fabrication, and inspection of microelements. Newly developed fringe projection 3D microscopy is presented in this paper. The system is configured according to camera-projector layout and long working distance lenses. The Scheimpflug principle is employed to make full use of the limited depth of field. For such a specific system, the general imaging model is introduced to reach a full 3D reconstruction. A dedicated calibration procedure is developed to realize quantitative 3D imaging. Experiments with a prototype demonstrate the accessibility of the proposed configuration, model, and calibration approach.

Prolonging life in chick forebrain-neuron culture and acquiring spontaneous spiking activity on a microelectrode array.

Various types of animal neurons were cultured on a microelectrode array (MEA) platform to form biosensors to detect potential environmental neurotoxins. For a large-scale screening tool, rodent MEA-based cortical-neuron biosensors would be very costly but chick forebrain neurons (FBNs) are abundant, cost-effective, and easy to dissect. However, chick FBNs have a lifespan of ~14 days in vitro and their spontaneous spike activity (SSA) has been difficult to develop and detect. We used a high-density neuron-glia co-culture on an MEA to prolong chick FBN lifetime to 3 months with lifetime-long SSA. A remarkable embryonic age-dependency in the culture's morphology, lifespan, and most features of SSA signal was discovered. Our results show the feasibility of developing a chick FBN-MEA biosensor and also establish a new electrophysiological platform for functional study of an in vitro neuronal network.

Enzyme-etching technique to fabricate micropatterns of aligned collagen fibrils.

A technique to tailor-make pre-coated, pre-aligned bovine collagen fibrils, derived from neonatal cardiomyocytes, on the surface of a glass slide into a designated pattern is reported. The unwanted collagen-coated area was erased by a collagenase solution and the tailored area was retained by attaching a microfabricated polydimethylsiloxane stamp directly to the collagen-coated surface. Using this technique, collagen patterns with designated orientations and with clear pattern boundaries and defined shapes were fabricated.

Measurement of the microscopic viscosities of microfluids with a dynamic optical tweezers system.

Viscosity coefficients of microfluids-Newtonian and non-Newtonian-were explored through the rotational motion of a particle trapped by optical tweezers in a microflute. Unlike conventional methods based on viscometers, our microfluidic system employs samples of less than 30 µl to complete a measurement. Viscosity coefficients of ethanol and fetal bovine serum, as typical examples of Newtonian and non-Newtonian fluids, were obtained experimentally, and found to be in excellent agreement with theoretical predictions. Additionally, a practical application to a DNA solution with incremental ethidium bromide content was employed and the results are consistent with clinical data, indicating that our system provides a potentially important complementary tool for use in such biological and medical applications.

Quantification of cardiac capillarization in basement-membrane-immunostained myocardial slices using Segment Anything Model.

Decreased myocardial capillary density has been reported as an important histopathological feature associated with various heart disorders. Quantitative assessment of cardiac capillarization typically involves double immunostaining of cardiomyocytes (CMs) and capillaries in myocardial slices. In contrast, single immunostaining of basement membrane protein is a straightforward approach to simultaneously label CMs and capillaries, presenting fewer challenges in background staining. However, subsequent image analysis always requires expertise and laborious manual work to identify and segment CMs/capillaries. Here, we developed an image analysis tool, AutoQC, for automatic identification and segmentation of CMs and capillaries in immunofluorescence images of basement membrane. Commonly used capillarization-related measurements can be derived from segmentation results. By leveraging the power of a pre-trained segmentation model (Segment Anything Model, SAM) via prompt engineering, the training of AutoQC required only a small dataset with bounding box annotations instead of pixel-wise annotations. AutoQC outperformed SAM (without prompt engineering) and YOLOv8-Seg, a state-of-the-art instance segmentation model, in both instance segmentation and capillarization assessment. Thus, AutoQC, featuring a weakly supervised algorithm, enables automatic segmentation and high-throughput, high-accuracy capillarization assessment in basement-membrane-immunostained myocardial slices. This approach reduces the training workload and eliminates the need for manual image analysis once AutoQC is trained.

Conditioned serum-free medium from umbilical cord mesenchymal stem cells has anti-photoaging properties.

Chronic exposure to solar radiation is the primary cause of photoaging and benign and malignant skin tumors. A conditioned serum-free medium (SFM) was prepared from umbilical cord mesenchymal stem cells (UC-MSCs) and its anti-photoaging effect, following chronic UV irradiation in vitro and in vivo, was evaluated. UC-MSC SFM had a stimulatory effect on human dermal fibroblast proliferation and reduced UVA-induced cell death. In addition, UC-MSC SFM blocked UVA inhibition of superoxide dismutase activity. Topical application of UC-MSC SFM to mouse skin prior to UV irradiation blocked the inhibition of superoxide dismutase and glutathione peroxidase activities, and prevented the upregulation of malonaldehyde. UC-MSC SFM thus protects against photoaging induced by UVA and UVB radiation and is a promising candidate for skin anti-photoaging treatments.

Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment.

Autofluorescence (AF) poses challenges for detecting proteins of interest in situ when employing immunofluorescence (IF) microscopy. This interference is particularly pronounced in strongly autofluorescent tissues such as myocardium, where tissue AF can be comparable to IF. Although various histochemical methods have been developed to achieve effective AF suppression in different types of tissue, their applications on myocardial  samples have not been well validated. Due to inconsistency across different autofluorescent structures in sometypes of tissue, it is unclear if these methods can effectively suppress AF across all autofluorescent structures within the myocardium. Here, we quantitatively evaluated the performance of several commonly used quenching treatments on formaldehyde-fixed myocardial samples, including 0.3 M glycine, 0.3% Sudan Black B (SBB), 0.1% and 1% sodium borohydride (NaBH4), TrueVIEW® and TrueBlack®. We further assessed their quenching performance by employing the pre-treatment and post-treatment protocols, designed to cover two common IF staining scenarios where buffers contained detergents or not. The results suggest that SBB and TrueBlack® outperform other reagents in AF suppression on formaldehyde-fixed myocardial samples in both protocols. Furthermore, we inspected the quenching performance of SBB and TrueBlack® on major autofluorescent myocardial structures and evaluated their influence on IF imaging. The results suggest that SBB outperforms TrueBlack® in quenching major autofluorescent structures, while TrueBlack® excels in preserving IF labeling signal. Surprisingly, we found the treatment of NaBH4 increased AF signal and enhanced the AF contrast of major autofluorescent structures. This finding suggests that NaBH4 has the potential to act as an AF enhancer and may facilitate the interpretation of myocardial structures without the need for counterstaining.

Strategy for automatic and complete three-dimensional optical digitization.

This Letter proposes a new strategy of a three-dimensional (3D) scanning pipeline to achieve complete 3D digitization of complex objects in a real scene. This strategy consists of a one-dimensional array of optical 3D sensors combined with an automatically controlled turntable. An efficient calibration method for the sensor array is presented to guarantee the accuracy of the 3D measurement. Furthermore, an automatic registration technique is also proposed for aligning multiple range images taken from sensor array. Experiment results are also presented to demonstrate the proposed approach.

Calibration of fringe projection profilometry with bundle adjustment strategy.

The measurement accuracy of fringe projection profilometry (FPP) largely depends on the calibration procedure. A more reliable calibration approach based on the stereo vision model of the FPP scheme in conjunction with the bundle adjustment strategy is presented. It can adjust the coordinates of benchmarks and thereby estimate the scheme parameters more accurately even with an imperfect target. The experiment results shows that the proposed approach can reach highly accurate calibration by solely using a printed target pattern, which verifies the proposed approach.

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator.

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 µm × 136 µm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.