Increasing the Maximum Detectable Flow Velocity in High-Frequency Ultrasound Vector Doppler Imaging.

High-frequency ultrasound (HFUS; >30 MHz) Doppler imaging has been widely used in the imaging of small animals and humans because of its high resolution. Vector Doppler imaging (VDI) has certain advantages for visualizing complex flow patterns independent of the Doppler angle. However, no commercial HFUS VDI system is currently available; therefore, several studies have connected an ultrasound research platform (Verasonics Vantage 256) with an HFUS array transducer for HFUS VDI. Unfortunately, the maximum frame rate of this system is only 10 kHz at an operational frequency of 40 MHz because of limitations related to data transmission hardware, thereby restricting the maximum detectable velocity of Doppler measurements. To address this drawback, in the present study, an electrocardiography (ECG)-gating-based HFUS VDI system was developed to avoid Doppler flow aliasing in data acquisition by ultrasound research platform at its maximum frame rate of 10 kHz. The developed method aligns all tilted plane waves with the ECG R-wave, which avoids the trade-off between frame rate and tilted angles number in conventional VDI. The performance of the proposed data acquisition method in HFUS VDI was verified using a steady-flow phantom, for which estimation errors were less than 10% under different flow settings. In animal studies, peak flow velocities in the carotid artery, left ventricle, and aortic arch of wild-type mice were measured (approximately 55, 655, and 765 mm/s, respectively). Also, the HFUS VDI from the mitral regurgitation mice model was obtained to present the complex flow patterns through the proposed method. In contrast to the conventional method, no Doppler aliasing occurs in the proposed method because the frame rate is sufficient. The experimental results indicate the developed HFUS VDI has the potential to become a useful tool for vector flow visualization in small animals, even under a high flow velocity.

High-resolution Tissue Doppler and Strain Imaging for Adult Zebrafish Myocardial Tissue through Ultrafast High-frequency Ultrasound Vector Doppler Estimation.

Zebrafish has been considered as an essential small-animal model for investigating the mechanism of heart regeneration. Due to the small size of zebrafish heart, high-frequency ultrasound (HFUS) imaging is often required for in vivo evaluations of its dynamic functions. Although commercial HFUS systems are available for myocardial velocity and strain measurement, only the outer myocardial region can be quantified due to the complex structure of zebrafish heart. In this study, a high-resolution 2D myocardial tissue Doppler and strain imaging based on ultrafast HFUS imaging was developed for zebrafish heart imaging during heart regeneration. The cardiac flow region was first extracted to recognize the myocardial region, and the myocardial velocity and strain were then determined through vector Doppler estimation. Adult AB-line zebrafish were used for in vivo experiments, and cryoinjury was induced in the apical region of the heart. Both the myocardial velocity and strain of the whole ventricle after cryoinjury were directly visualized over 28 days. Myocardial velocity (during later diastolic motion) and strain, respectively, were significantly decreased (anterior wall: -2.0 mm/s and -3.3%; apical region: -2.0 mm/s and -4.5%; posterior wall: -1.7 mm/s and -4.3%) at the first 3 days after cryoinjury, which indicates weak myocardial beating due to heart injury. However, these all returned to the baseline values at 14 days after cryoinjury. All of the experimental results indicate that the proposed method is a useful tool for heart regeneration studies in adult zebrafish. In particular, it allows for the noninvasive evaluation of regional dynamic heart function.

A gated recurrent unit model based on ultrasound images of dynamic tongue movement for determining the severity of obstructive sleep apnea.

Obstructive sleep apnea (OSA) presents as a respiratory disorder characterized by recurrent upper pharyngeal airway collapse during sleep. Dynamic tongue movement (DTM) analysis emerges as a promising avenue for elucidating the pathophysiological underpinnings of OSA, thereby facilitating its diagnosis. Recent endeavors have utilized artificial intelligence techniques to categorize OSA severity leveraging electrocardiography and blood oxygen saturation data. Nonetheless, the integration of ultrasound (US) imaging of the tongue remains largely untapped in the development of machine learning models aimed at determining the severity of OSA. This study endeavors to bridge this gap by capturing US images of DTM dynamics during wakefulness, encompassing transitions from normal breathing (NB) to the performance of the Müller maneuver (MM) in a cohort of 53 patients. Leveraging the modified optical flow method (MOFM), the trajectories of patients' DTM were tracked, facililtating the extraction of 27 parameters vital for model training. These parameters encompassed nine-point lateral movement, nine-point axial movement, and nine-point total displacement of the tongue, resulting in a dataset of 186,030 samples. The gated recurrent unit (GRU) method, renowned for its efficacy in motion tracking, was employed for model development in this study. Validation of the developed model was conducted via stratified k-fold cross-validation (SCV). The systems' overall performance in classifying OSA severity, as quantified by mean accuracy (MA), yielded a value of 43.49%. This pilot investigation marks an exploratory endeavor into the utilization of artificial intelligence for the classification of OSA severity based on US images and dynamic movement patterns. This novel model holds potential to assist clinicians in categorizing OSA severity and guiding the selection of pertinent treatment modalities tailored to the individual needs of patients afflicted with OSA.

High-Performance PMN-PT Single-Crystal-Based 1-3 Composite Transducer Integrated with a Biopsy Needle.

To address the need for high-resolution imaging in lung nodule detection and overcome the limitations of the shallow imaging depth associated with high-frequency ultrasound and the complex structure of lung tissue, we successfully integrated 50 MHz ultrasound transducers with 18-gauge biopsy needles. Featuring a miniaturized size of 0.6 × 0.5 × 0.5 mm3, the 50 MHz micromachined 1-3 composite transducer was tested to perform mechanical scanning of a nodule within a lung-tissue-mimicking phantom in vitro. The high-frequency transducer demonstrated the ability to achieve imaging with an axial resolution of 30 µm for measuring nodule edges. Moreover, the integrated biopsy needle prototype exhibited high accuracy (1.74% discrepancy) in estimating nodule area compared to actual dimensions in vitro. These results underscore the promising potential of biopsy-needle-integrated transducers in enhancing the accuracy of endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNA) for clinical applications.

Wearable Ultrasound Imaging Device for Dynamic Dual-Direction Shear Wave Elastography of Shoulder Muscle.

The shoulder is the most mobile joint in the human body, thus requiring intricate coordination of adjacent muscles. Patients suffered from rotator cuff muscle injuries have several typical symptoms including shoulder pain and difficulty raising the arm, thus reducing work efficiency and compromising the quality of life. Ultrasound has been used widely for shoulder soft tissue imaging as well as ultrasound elastography was introduced in shoulder examination for the dilemma of treating degenerative rotator cuff tears. However, most of the ultrasound examination was performed under a static condition. Providing dynamic information from shoulder muscle is important in clinical applications because the pains sometimes come from various positions of the shoulder during moving. In this study, a customized wearable T-shaped ultrasound transducer (128 + 128 elements) was proposed for shoulder dual-direction shear wave elastography (DDSWE), which provides the SWE for both longitudinal (SW along the muscle fiber) and transverse (SW cross the muscle fiber) directions dynamically. An optical tracking system was synchronized with an ultrasound imaging system to capture shoulder movements in 3-D space with their corresponding ultrasound images. The performance of DDSWE and the accuracy of optical tracking were verified by phantom experiments. Human studies were carried out by volunteers as they are moving their arms. The experimental results show that the bias and precision for the proposed DDSWE in elastic phantom were about 6% and 1.2% for both directions, respectively. A high accuracy of optical tracking was observed using a 3-D motor stage experimental setup. Human experiments show that the shear wave velocities (SWVs) were increased with the angles of shoulder abduction, and the average transverse and longitudinal SWVs were increased from 2.24 to 3.35 m/s and 2.95 to 5.95 m/s with abduction angle from 0° to 60°, respectively, which they are anisotropic-dependent. All the experimental results indicate that the proposed wearable ultrasound DDSWE can quantify the mechanical properties of shoulder muscles dynamically, thereby helping surgeons and physical therapists determine whether the intensity of rehabilitation shoulder be tuned down or escalated in the future.

High-Spatiotemporal-Resolution Ultrasound Flow Imaging to Determine Cerebrovascular Hemodynamics in Alzheimer's Disease Mice Model.

Although the relationships of cerebrovascular hemodynamic dysfunction with neurodegenerative diseases remain unclear, many studies have indicated that poor cerebral perfusion accelerates the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). Small animal models are widely used in AD research. However, providing an imaging modality with a high spatiotemporal resolution and sufficiently large field of view to assess cerebrovascular hemodynamics in vivo remains a challenge. The present study proposes a novel technique for high-spatiotemporal-resolution vector micro-Doppler imaging (HVµDI) based on contrast-free ultrafast high frequency ultrasound imaging to visualize the cerebrovascular hemodynamics of the mouse, with a data acquisition time of 0.4 s, a minimal detectable vessel size of 38 µm, and a temporal resolution of 500 Hz. In vivo experiments are conducted on wild-type and AD mice. Cerebrovascular hemodynamics are quantified using the cerebral vascular density, diameter, velocity, tortuosity, cortical flow pulsatility, and instant flow direction variations. Results reveal that AD significantly change the cerebrovascular hemodynamics. HVµDI offers new opportunities for in vivo analysis of cerebrovascular hemodynamics in neurodegenerative pathologies in preclinical animal research.

Visualization of Human Hand Tendon Mechanical Anisotropy in 3-D Using High- Frequency Dual-Direction Shear Wave Imaging.

High-resolution ultrasound shear wave elastography has been used to determine the mechanical properties of hand tendons. However, because of fiber orientation, tendons have anisotropic properties; this results in differences in shear wave velocity (SWV) between ultrasound scanning cross sections. Rotating transducers can be used to achieve full-angle scanning. However, this technique is inconvenient to implement in clinical settings. Therefore, in this study, high-frequency ultrasound (HFUS) dual-direction shear wave imaging (DDSWI) based on two external vibrators was used to create both transverse and longitudinal shear waves in the human flexor carpi radialis tendon. SWV maps from two directions were obtained using 40-MHz ultrafast imaging at the same scanning cross section. The anisotropic map was calculated pixel by pixel, and 3-D information was obtained using mechanical scanning. A standard phantom experiment was then conducted to verify the performance of the proposed HFUS DDSWI technique. Human studies were also conducted where volunteers assumed three hand postures: relaxed (Rel), full fist (FF), and tabletop (TT). The experimental results indicated that both the transverse and longitudinal SWVs increased due to tendon flexion. The transverse SWV surpassed the longitudinal SWV in all cases. The average anisotropic ratios for the Rel, FF, and TT hand postures were 1.78, 2.01, and 2.21, respectively. Both the transverse and the longitudinal SWVs were higher at the central region of the tendon than at the surrounding region. In conclusion, the proposed HFUS DDSWI technique is a high-resolution imaging technique capable of characterizing the anisotropic properties of tendons in clinical applications.

Visualization of Pulse-Wave Velocity on Arterial Wall of Mice Through High-Frequency Ultrafast Doppler Imaging.

Arterial pulse-wave velocity (PWV) is widely used in clinical applications to assess cardiovascular diseases. Ultrasound methods have been proposed for estimating regional PWV in human arteries. Furthermore, high-frequency ultrasound (HFUS) has been applied to perform preclinical small-animal PWV measurements; however, electrocardiogram (ECG)-gated retrospective imaging is required to achieve high-frame-rate imaging, which might be affected by arrhythmia-related problems. In this article, HFUS PWV mapping based on 40-MHz ultrafast HFUS imaging is proposed to visualize PWV on mouse carotid artery to measure arterial stiffness without ECG gating. In contrast to most other studies that used cross-correlation methods to detect arterial motion, ultrafast Doppler imaging was applied in this study to measure arterial wall velocity for PWV estimations. The performance of the proposed HFUS PWV mapping method was verified using a polyvinyl alcohol (PVA) phantom with various freeze-thaw cycles. Small-animal studies were then performed in wild-type (WT) mice and in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (for 16 and 24 weeks). The Young's modulus of the PVA phantom measured through HFUS PWV mapping was 15.3 ± 0.81, 20.8 ± 0.32, and 32.2 ± 1.11 kPa for three, four, and five freeze-thaw cycles, respectively, and the corresponding measurement biases (relative to theoretical values) were 1.59%, 6.41%, and 5.73%, respectively. In the mouse study, the average PWVs were 2.0 ± 0.26, 3.3 ± 0.45, and 4.1 ± 0.22 m/s for 16-week WT, 16-week ApoE KO, and 24-week ApoE KO mice, respectively. The PWVs of ApoE KO mice increased during the high-fat diet feeding period. HFUS PWV mapping was used to visualize the regional stiffness of mouse artery, and a histology confirmed that the plaque formation in the bifurcation region increased the regional PWV. All the results indicate that the proposed HFUS PWV mapping method is a convenient tool for investigating arterial properties in preclinical small-animal studies.

Using a Flipped Classroom to Compare 2 Ultrasonography Operating Methods to Improve Practice in Ultrasound Detection Acupuncture.

Objective: Ultrasound (US) detection acupuncture (UDA) is an innovative acupuncture technique that uses ultrasonography (USG) to detect the depth of the lung before performing acupuncture on the points around the chest to avoid puncturing the lungs. For acupuncturists to use UDA appropriately, it is crucial to have a good operating method to identify the pleura with USG. This study compared 2 US operating methods through active learning in a "flipped classroom" setting for acupuncture students. Materials and Methods: Students and interns were recruited to complete the UDA flipped classroom course and evaluate the operations of 2 US methods on either of 2 simulation models: (1) a single B-mode or (2) a combined M-mode + B-mode. Participants were interviewed and satisfaction surveys were administered to obtain feedback. Results: A total of 37 participants completed the course and evaluations. The combined mode had better measurement accuracy, acupuncture safety, and operating time (P < 0.05), and no pneumothoraxes occurred. Among both participant groups, the combined mode allowed the student group to learn quickly and the intern group to become more proficient. Both interviews and satisfaction surveys yielded positive feedback. Conclusions: Using a combined mode for UDA can improve its performance greatly. The combined mode is definitely helpful for learning and promotion of UDA.

Estimation of Mouse Carotid Arterial Wall Shear Stress Using High-Frequency Ultrasound Imaging.

Wall shear stress (WSS) is a crucial hemodynamic factor that promotes atherosclerosis (plaque) development in arteries; although the relationship between WSS and arterial atherosclerosis has been explored in many animal studies, it is not fully understood. No suitable tool, however, exists for rapidly estimating dynamic WSS in small-animal studies. This study proposes a 40-MHz high-frequency ultrasound (HFUS) imaging system for dynamic WSS estimation based on mouse carotid artery blood flow velocity gradient measurements by vector Doppler imaging (VDI). Aliasing reduces the accuracy of Doppler measurements, which can be prevented by increasing the imaging frame rate. Conventionally, imaging is performed at two tilted angles by alternating between the angles; in the proposed method, the frame rate was doubled by imaging at each tilted angle sequentially and by then temporally aligning the sequences based on pulsatile flow characteristics. Velocity estimation using this method had low errors for both a steady-flow straight-tube and pulsatile flow 60%-stenosis phantom. The method was tested for wild-type (WT) C57BL/6 mice at 16 weeks old and apolipoprotein E knockout (ApoE KO) mice at 16 and 24 weeks old; differences in time-averaged and oscillatory WSS were observed, and histology confirmed that the 24-week ApoE KO mice with the highest oscillatory WSS had the greatest plaque formation. The proposed HFUS WSS imaging method can predict the location and extent of plaque development; thus, this method is useful for small-animal studies investigating the WSS effect on atherosclerotic plaque development.

Multifunctional Hydrogel Dressing That Carries Three Antibiotics Simultaneously and Enables Real-Time Ultrasound Bacterial Colony Detection.

We have developed a multifunctional hydrogel that can carry three synergistic antibiotics commonly used in clinical practice. This hydrogel was discovered to have drug encapsulation efficiencies of 94% for neomycin, 97% for bacitracin, and 88% for polymyxin B. Drug release data indicated that the release profiles of these three antibiotics were different. A swelling test demonstrated that the hydrogel absorbed liquid after the release of its antibiotics until it became saturated, which occurred within 48 h. Moreover, this hydrogel exhibited excellent antibacterial effects against Escherichia coli and Pseudomonas aeruginosa and biocompatibility; it can thus protect a wound from microbial invasion. When the alginate hydrogel is used to cover a wound, the wound can be checked for colonization at any time using ultrasound imaging; this can thus enable the prevention of wound biofilm formation in the early stages of infection. We evaluated the hydrogel against commercially available wound dressings and discovered that these wound dressings did not have the aforementioned desirable features. In conclusion, our multifunctional hydrogel can carry three types of antibiotics simultaneously and is a suitable medium through which an ultrasound can be performed to detect the growth of colonies in wounds. The hydrogel is expected to make a valuable contribution to the prevention of wound infections in the future.

High-frequency ultrasound imaging for monitoring the function of meningeal lymphatic system in mice.

The meningeal lymphatic system drains the cerebrospinal fluid from the subarachnoid space to the cervical lymphatic system, primarily to the deep cervical lymph nodes. Perturbations of the meningeal lymphatic system have been linked to various neurologic disorders. A method to specifically monitor the flow of meningeal lymphatic system in real time is unavailable. In the present study, we adopted the high-frequency ultrasound (HFUS) with 1,1'diocatadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-loaded microbubble and FePt@PLGA nanoparticle contrast agents to evaluate the flow of the meningeal lymphatic system in 2-month-old mice. Statistical analysis was performed to identify changes of HFUS signals among the microbubbles, FePt@PLGA nanoparticles, and saline control groups. Approximately 15 min from the start of intracerebroventricular injection of contrast agents, their signals were evident at the deep cervical lymph nodes and lasted for at least 60 min. These signals were validated on the basis of the presence of DiI and Fe signals in the deep cervical lymph nodes. Ligation of afferent lymphatic vessels to the deep cervical lymph nodes eliminated the HFUS signals. Moreover, ablation of lymphatic vessels near the confluence of sinuses decreased the HFUS signals in the deep cervical lymph nodes. Glioma-bearing mice that exhibited reduced lymphatic vessel immunostaining signals near the confluence of sinuses had lowered HFUS signals in the deep cervical lymph nodes within 60 min. The proposed method provides a minimally invasive approach to monitor the qualities of the meningeal lymphatic system in real time as well as the progression of the meningeal lymphatic system in various brain disease animal models.

Development of a Wearable Ultrasound Transducer for Sensing Muscle Activities in Assistive Robotics Applications.

Robotic prostheses and powered exoskeletons are novel assistive robotic devices for modern medicine. Muscle activity sensing plays an important role in controlling assistive robotics devices. Most devices measure the surface electromyography (sEMG) signal for myoelectric control. However, sEMG is an integrated signal from muscle activities. It is difficult to sense muscle movements in specific small regions, particularly at different depths. Alternatively, traditional ultrasound imaging has recently been proposed to monitor muscle activity due to its ability to directly visualize superficial and at-depth muscles. Despite their advantages, traditional ultrasound probes lack wearability. In this paper, a wearable ultrasound (US) transducer, based on lead zirconate titanate (PZT) and a polyimide substrate, was developed for a muscle activity sensing demonstration. The fabricated PZT-5A elements were arranged into a 4 × 4 array and then packaged in polydimethylsiloxane (PDMS). In vitro porcine tissue experiments were carried out by generating the muscle activities artificially, and the muscle movements were detected by the proposed wearable US transducer via muscle movement imaging. Experimental results showed that all 16 elements had very similar acoustic behaviors: the averaged central frequency, -6 dB bandwidth, and electrical impedance in water were 10.59 MHz, 37.69%, and 78.41 Ω, respectively. The in vitro study successfully demonstrated the capability of monitoring local muscle activity using the prototyped wearable transducer. The findings indicate that ultrasonic sensing may be an alternative to standardize myoelectric control for assistive robotics applications.

Numerical and experimental evaluation of low-intensity transcranial focused ultrasound wave propagation using human skulls for brain neuromodulation.

BACKGROUND: Low-intensity transcranial focused ultrasound (tFUS) has gained considerable attention as a promising noninvasive neuromodulatory technique for human brains. However, the complex morphology of the skull hinders scholars from precisely predicting the acoustic energy transmitted and the region of the brain impacted during the sonication. This is due to the fact that different ultrasound frequencies and skull morphology variations greatly affect wave propagation through the skull. PURPOSE: Although the acoustic properties of human skull have been studied for tFUS applications, such as tumor ablation using a multielement phased array, there is no consensus about how to choose a single-element focused ultrasound (FUS) transducer with a suitable frequency for neuromodulation. There are interests in exploring the magnitude and dimension of tFUS beam through human parietal bone for modulating specific brain lobes. Herein, we aim to investigate the wave propagation of tFUS on human skulls to understand and address the concerns above. METHODS: Both experimental measurements and numerical modeling were conducted to investigate the transmission efficiency and beam pattern of tFUS on five human skulls (C3 and C4 regions) using single-element FUS transducers with six different frequencies (150-1500 kHz). The degassed skull was placed in a water tank, and a calibrated hydrophone was utilized to measure acoustic pressure past it. The cranial computed tomography scan data of each skull were obtained to derive a high-resolution acoustic model (grid point spacing: 0.25 mm) in simulations. Meanwhile, we modified the power-law exponent of acoustic attenuation coefficient to validate numerical modeling and enabled it to be served as a prediction tool, based on the experimental measurements. RESULTS: The transmission efficiency and -6 dB beamwidth were evaluated and compared for various frequencies. An exponential decrease in transmission efficiency and a logarithmic decrease of -6 dB beamwidth with an increase in ultrasound frequency were observed. It is found that a >750 kHz ultrasound leads to a relatively lower tFUS transmission efficiency (<5%), whereas a <350 kHz ultrasound contributes to a relatively broader beamwidth (>5 mm). Based on these observations, we further analyzed the dependence of tFUS wave propagation on FUS transducer aperture size. CONCLUSIONS: We successfully studied tFUS wave propagation through human skulls at different frequencies experimentally and numerically. The findings have important implications to predict tFUS wave propagation for ultrasound neuromodulation in clinical applications, and guide researchers to develop advanced ultrasound transducers as neural interfaces.

High-frequency noncontact low-intensity pulsed ultrasound modulates Ca2+-dependent transcription factors contributing to cell migration.

Chronic wounds have negative physical and psychological effects on patients and increase the health care burden. Consequently, chronic wound in the elderly population is an important issue. Ultrasound can be a great modality for treating chronic wounds because of its noninvasive and safety characteristics; it can accelerate in vitro and in vivo wound healing. In this study, we developed a novel noncontact ultrasound for wound treatment. We stimulated human epidermal keratinocyte migration using low-intensity pulsed ultrasound (LIPUS) with a noncontact transducer to avoid direct contact with the wound. We also compared the effects of 15-min contact and noncontact transducer stimulation, where a 1-MHz contact transducer (intensity = 40 or 200 mW/cm2) and a 0.45-MHz noncontact transducer (intensity = 30 mW/cm2) were used. Both contact and noncontact LIPUS considerably increased cell migration and activated the calcium (Ca2+)-dependent transcription factors cAMP-responsive element-binding protein (CREB) and nuclear factor of activated T cells (NFAT). Furthermore, noncontact transducer stimulation did not cause cell death or affect cell proliferation but significantly increased the Ca2+ influx-mediated intracellular Ca2+ levels. Ca2+-free medium and Ca2+ channel blockers effectively inhibited LIPUS-induced Ca2+-dependent transcription factor activation and cell migration.

Intermittent theta burst stimulation for the treatment of autonomic dysfunction and depressive symptoms in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is one of the most prevalent forms of neurodegenerative dementia, second to Alzheimer's disease, and autonomic abnormalities and depressive symptoms are common. There are currently no cures or treatments with evidence of disease-modifying effects for DLB, and the treatment for the amelioration of targeted symptoms is challenging due to the risk of side effects and drug-drug interactions. In the present case, we report a female elder with DLB suffering from poor tolerance to the adverse events of numerous approaches. Following intermittent theta burst stimulation (iTBS), the autonomic abnormalities and depressive symptoms remarkably improved without significant side effects.

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

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