Neural tube closure (NTC) is a highly synchronized morphological process driven by mechanical forces, and any disruptions during this process can lead to neural tube defects (NTDs). However, mechanical properties associated with NTDs are largely unknown. To understand the correlation between NTDs and biomechanical properties, we imaged NTC using multimodal Brillouin microscopy and optical coherence tomography in two mutant mice lines, where the genes Mthfd1l and Fuz were inactivated. We also imaged cerebral organoids cultured in dolutegravir for 10 and 14 days. Our results showed a clear link between NTDs and neural tube biomechanical properties.
Many embryonic developmental processes are inherently mechanical, such as elongation, neural tube closure, and cardiogenesis. Any disruption or failure of these events can lead to debilitating or even fatal pathologies, e.g., anencephaly. While much is known about the genetic and molecular mechanisms underlying these processes, there remains a significant knowledge gap about the associated biomechanical parameters due to the lack of noninvasive high-resolution mechanical imaging techniques, particularly in live samples. In this work, we demonstrate completely noninvasive, label-free, high-resolution, and three-dimensional mapping of mouse embryo stiffness at several critical stages of embryogenesis based on reverberant shear wave optical coherence elastography (Rev-OCE). Mouse embryos at various developmental stages (embryonic day 9.5, 10.0, 10.5, 11.0, and 11.5) were dissected out and placed on an optical window during imaging. The samples were encompassed in embryo culture media to preserve the integrity of the delicate embryo tissues. The optical window was attached to a piezoelectric bender, which vibrated the optical window at 1kHz. M-C-mode imaging was performed with a phase-sensitive spectral domain OCT system operating in the common-path configuration. Standard reverberant OCE processing steps were applied, and the local autocorrelation was fitted to the analytical solution of the reverberant shear field. The local shear wave speed was then mapped in 3D. The results show that the stiffness of the spine, heart, and brain all increased as the embryo developed.
Phenylthiourea (PTU) is often used to block pigmentation and make zebrafish completely transparent for easy optical imaging. PTU inhibits melanogenesis by inhibiting tyrosinase. Although the PTU is commonly used, it does have some side effects. PTU at a concentration of 0.2 mM (0.003%) significantly reduces the zebrafish eye size due to the inhibition of thyroid hormone production. Furthermore, low levels of thyroid hormones in zebrafish increase the stiffness of the intervertebral joints, altering their swimming behavior. The aim of this study was to assess the structural modifications and biomechanical properties of 5-day post-fertilization (dpf) zebrafish eyes after being exposed to PTU using optical coherence tomography and reverberant optical coherence elastography, respectively. Wild-type zebrafish (n=3), treated with PTU (0.2 mM), were compared with non-treated zebrafish (n=3). The results show a significant reduction (p=0.02) in the mean eye diameter of the fishes treated with PTU (312.66 ± 8.71 μm) versus the non-treated group (340.18 ± 4.38 μm). On the other hand, the non-treated group showed a significantly slower (p=0.02) shear wave speed (0.97 ± 0.12 m/s) compared with the PTU-treated group (2.65 ± 0.51 m/s), indicating that PTU induces a biomechanical change in the stiffness of the developing eye. PTU is a potent inhibitor of the pigmentation of zebrafish; however, it can also severely affect its biomechanical properties, specifically eye development, reducing eye diameter and increasing its stiffness.
Measuring the mechanical properties of the cornea can help understand the structure and physiology of the eye, early detection of disease, and evaluation of therapy outcomes. In this work, we investigate the effect of collagen XII deficiency on the stiffness of the murine cornea using a multimodal approach for biomechanical analysis. Wave-based optical coherence elastography (OCE), heartbeat OCE, and Brillouin microscopy were all utilized to assess the mechanical properties of wild-type and collagen XII deficient ex vivo murine corneas as a function of IOP. All three techniques show that collagen XII deficiency leads to a dramatic decrease in corneal stiffness. Future work will investigate how these measurement techniques can be translated for in vivo assessment of corneal elasticity to understand the contribution of various proteins to corneal structural and mechanical integrity.
Embryo development is driven by several substantial events that cause structural modifications during growth. The progressive changes in the embryo during this important period cause simultaneous alterations of its biomechanical properties. Understanding the structural modifications and changes in stiffness during embryo development is important for comprehending its growth and potentially detecting congenital diseases. The aim of this study is to map the biomechanical properties of embryos in 3D during development. Murine embryos at gestational day (GD) 11 were imaged using 3D Reverberant optical coherence elastography (Rev-OCE). The embryos were placed on a glass window, which was vibrated by an actuator at 1 kHz. In addition to providing the vibration, the glass window also enabled imaging in the common path configuration, which eliminated environmental noise and improved the displacement sensitivity of the system to sub-nanometer levels. The results showed the structural changes and the differences in stiffness in the embryo. The stiffness of the embryos from GD 11 showed stiffer areas along the developing spinal cord. Combining high-resolution OCT with elastography allowed us to understand the structural and biomechanical changes in the embryo during its development. Thus, this study provides important insights into embryo mechanical properties, which could serve as a potential biomarker for deficiencies in embryo development. Our future work is focused on imaging embryos at different stages as well as studying mutant models of congenital diseases, such as neural tube defects.
Neural tube closure is a complex process driven by mechanical forces, but this process can be disturbed leading to development defects. So, to understand the interplay between forces and tissue stiffness during neurulation, we developed a multimodal Brillouin microscopy and optical coherence system (OCT). OCT provides structural guidance while mapping the biomechanical properties of embryonic neural tube using Brillouin microscopy. 3D-OCT, 2D-OCT, and 2D-Brillouin images of Mthfd1l and Fuz knockout mouse embryos at gestation days 9.5 and 10.5 were acquired. Our results show overall decrease in the stiffness of homozygotic knockout neural tube tissues compared to the wildtype.
The heart is the first essential organ that develops during organogenesis. Fetal impaired heart function correlates to functional cardiac anomalies and heart defects in adulthood. Therefore, noninvasive assessment of dynamic functional cardiac events during pregnancy is essential for early diagnosis of cardiac diseases. However, visualization and analysis of the small yet fast-beating embryonic heart require a high-resolution imaging platform to provide reliable volumetric analyses. Optoacoustic (OA) imaging provides excellent optical contrast along with high spatial resolution and has demonstrated an exclusive potential for noninvasive deep-tissue visualization. In this study, we used volumetric OA imaging to visualize the embryonic heart at gestational day (GD) 16.5. The anatomical structure of the embryonic heart and cardiac vasculature was visualized in three orthogonal imaging planes allowing for further quantification and structural measurements. Twenty-five volumes per second temporal resolution of OA imaging enabled assessment of embryonic cardiac dynamics. Using the temporal profile of the time-lapse OA data at different locations of the embryonic heart, the average heart rate of embryos was calculated. This study demonstrated the capability of volumetric OA tomography for noninvasive visualization of the embryonic heart and assessment of cardio dynamics at nearly video rate.
The healthy development of embryos depends on several critical biomechanical processes, such as neurulation and the formation of the cardiovascular system. Thus, understanding the structural modifications and changes in stiffness during development is important for understanding the etiology of various congenital diseases, such as anencephaly or spina bifida. In this work, we demonstrate the ability of reverberant optical coherence elastography (Rev-OCE) to map the biomechanical properties of various small animal embryos in high resolution in 3D completely noninvasively and without the need for any exogenous contrast agents. Rev-OCE measurements were performed in both murine and zebrafish embryos to showcase its capability to map the stiffness of commonly used small animal models of disease. The murine embryos were dissected from CD1 mice at gestational day 11, and the zebrafish embryos were isolated at 7 days post fertilization. Rev-OCE imaging was performed using a phase-sensitive optical coherence tomography (PhS-OCT) system, where the samples were placed on a glass window that was attached to a piezoelectric bender. The bender vibrated and generated randomly oriented shear waves in the samples, which were detected by the PhS-OCT system. In addition to holding the samples, the glass window enabled common path imaging for sub-nanometer levels of displacement sensitivity. The results show a clear spatial distribution of stiffness in the embryos. For example, the spinal region of the murine embryos was stiffer than other tissues, and in the zebrafish embryos, the head and swim bladder were stiffer. Embryonic elasticity could provide valuable insight into the critical embryonic developmental process and etiology of various congenital defects.
Fluorescent two-photon selective-plane illumination microscopy (2P-SPIM) enables deep imaging of cellular information such as proliferation, type identification, and signaling using fluorescence. Optical coherence tomography (OCT) can capture complementary structural information based on intrinsic optical scattering. We developed a specialized multimodal high-resolution embryonic imaging system combining the benefits of OCT with 2P-SPIM. The OCT and 2P-SPIM beams were optically co-aligned and scanned using the same scanners and the same objective lens. The resulting light sheet thickness was ~13 µm with a transverse resolution of ~2.1 µm. The OCT system was based on a 1050 nm centered swept source laser with a bandwidth of ~100 nm and a sweep rate of 100 kHz. The OCT system utilized a Michelson-style interferometer and had a lateral resolution of ~15 µm and an axial resolution of ~7 µm. The capabilities of the multimodal imaging system were demonstrated using images of fluorescent microbeads and a fluorescently tagged mouse embryo at gestational day 9.5. Due to the co-alignment of the OCT and 2P-SPIM systems, image registration was simple and allowed for high-throughput multimodal imaging without the use of sophisticated registration methods.
The mechanisms involved in neural tube formation are complex and can be easily disrupted. Neurulation is one such process, governed by mechanical forces where tissues physically fold and fuse. When neural tube folding and closure fail to complete during neurulation, it results in structural and functional abnormalities of the brain and spinal cord. Thus, it is important to understand the interplay between forces and tissue stiffness during neurulation. Brillouin microscopy is an all-optical, noninvasive, high-resolution imaging technique capable of mapping tissue stiffness, but it cannot provide structural information, resulting in “blind” imaging. To overcome this limitation, we have combined a Brillouin microscopy system with optical coherence tomography (OCT) in one synchronized and co-aligned instrument to provide structural guidance when mapping the biomechanical properties of neural tube formation in mouse embryos. We developed custom instrumentation control software that utilizes the OCT structural image to guide Brillouin imaging. We acquired first 3D OCT images and then 2D structural and mechanical maps of mouse embryos at embryonic day (E) 8.5, 9.5, and 10.5. Brillouin microscopy showed the cell-dense layer of neural plate derived from the ectoderm at E 8.5, which was unable to be distinguished with OCT. At E 9.5 and 10.5, the neuroepithelium could be clearly seen by Brillouin microscopy with a greater stiffness than the surrounding tissue. Our results show the capability of the co-aligned and synchronized Brillouin-OCT system to map tissue stiffness of murine embryos using OCT-guided Brillouin microscopy.
Optical coherence tomography (OCT) and light sheet fluorescence microscopy (LSFM) are well-established imaging techniques preferred in developmental biology, e.g., embryonic imaging. However, each technique has its own drawbacks, such as resolution and molecular specificity with OCT and field-of-view (FOV) and speed with LSFM. To overcome these limitations for small animal embryo imaging, we have developed a co-aligned multimodal imaging system combining OCT and LSFM. The OCT probe and LSFM excitation beams were combined and scanned with a galvanometer-mounted mirror through the same objective lens. The light sheet thickness was ~13 μm. The LSFM collection arm consisted of a 0.8 numerical aperture water immersion objective, tube lens, and CCD camera, resulting in a transverse resolution of ~2.1 μm. The OCT system was based on a 100 kHz swept-source laser with a central wavelength of 1050 nm and had a lateral resolution of ~15 µm and an axial resolution of ~7 μm. Images of fluorescent microbeads and a fluorescent-tagged mouse embryo at gestational day 9.5 showed the capabilities of the multimodal imaging system. Since the OCT system and LSFM system were co-aligned, image registration was straightforward and enabled high-throughput multimodal imaging without the need for complex registration techniques.
Retinal diseases such as diabetic retinopathy and glaucoma are leading cause of blindness in the world. Assessing biomechanical properties of retina is very crucial since it is constantly under stress due to the vitreous humor and eye movements. Characterizing biomechanical properties of retina noninvasively is a challenge due to its location inside the eye-globe, fragility, and thin geometry. Brillouin microscopy is a noninvasive, all optical imaging technique to qualitatively map the biomechanical properties of tissues. In this work, we mapped the layer by layer distribution of biomechanical properties of retinas using Brillouin microscopy. We found that the nuclear layer was stiffer compared to other layers. Furthermore, we observed fixing the retinas with paraformaldehyde increased the retinal stiffness compared to the fresh retinas.
In this study, we propose to measure the spatial deformation spreading (SDS) produced by an air-pulse on the surface of tissues during the near-field regime of propagation as a metric to characterize degree of anisotropy. Experiments in isotropic tissue-mimicking phantoms and anisotropic chicken tibialis muscle were conducted using an optical coherence tomography system synchronized with a confocal air-pulse stimulation. SDS ratio measured along versus across the direction of fibers in chicken muscle agreed with the wave speed ratio taken at the same directions, demonstrating the capabilities of the air-pulse SDS technique in measuring the elastic anisotropy of transverse isotropic tissues.
We have designed and developed an air-coupled ultrasonic radiation force probe co-focused with a phase-sensitive optical coherence tomography system (ACUS-OCT). Our custom-made 1 MHz spherically focused piezo-electric transducer with a concentric circular opening of 10 mm diameter allows for the confocal micro-excitation of waves and the spatial (2D, 3D) motion measurement of tissues. Phantom studies demonstrated the capabilities of this probe to produce quasi-harmonic excitation up to 4 kHz for the generation of highly localized elastic waves. Experimental results in ocular tissues showed the highly localized 2D/3D elasticity mapping capabilities of this approach with great potential for clinical translation.
Significance: The retina is critical for vision, and several diseases may alter its biomechanical properties. However, assessing the biomechanical properties of the retina nondestructively is a challenge due to its fragile nature and location within the eye globe. Advancements in Brillouin spectroscopy have provided the means for nondestructive investigations of retina biomechanical properties.
Aim: We assessed the biomechanical properties of mouse retinas using Brillouin microscopy noninvasively and showed the potential of Brillouin microscopy to differentiate the type and layers of retinas based on stiffness.
Approach: We used Brillouin microscopy to quantify stiffness of fresh and paraformaldehyde (PFA)-fixed retinas. As further proof-of-concept, we demonstrated a change in the stiffness of a retina with N-methyl-D-aspartate (NMDA)-induced damage, compared to an undamaged sample.
Results: We found that the retina layers with higher cell body density had higher Brillouin modulus compared to less cell-dense layers. We have also demonstrated that PFA-fixed retina samples were stiffer compared with fresh samples. Further, NMDA-induced neurotoxicity leads to retinal ganglion cell (RGC) death and reactive gliosis, increasing the stiffness of the RGC layer.
Conclusion: Brillouin microscopy can be used to characterize the stiffness distribution of the layers of the retina and can be used to differentiate tissue at different conditions based on biomechanical properties.
One of the most common reasons of congenital birth defects is prenatal substance abuse. The severity of the defect depends on the amount of substance abused and the period of gestation during which substance is abused. Although prenatal substance abuse is common during the first trimester, some women continue the abuse well into their second trimester, which is considered the peak period for fetal neurogenesis and angiogenesis. Thus, evaluating the changes in fetal brain vasculature caused by maternal exposure to different teratogens at the second trimester equivalent period is crucial. In this study we use correlation mapping optical coherence angiography (cm-OCA), a functional extension of optical coherence tomography, to image changes in murine fetal brain vasculature caused due to prenatal exposure to ethanol, cannabinoids, or nicotine. Results showed significant vasoconstriction in all three cases.
The biomechanical properties of the crystalline lens play a crucial role in its visual function. Assessing biomechanical properties of the lens may help with early disease detection and robust assessment of therapeutic interventions. However, measuring the biomechanical properties of the lens is a challenge due to its location inside the eye-globe. In this study, we demonstrate the combination of optical coherence elastography (OCE) and Brillouin microscopy to evaluate the stiffness of porcine lenses ex vivo (N=6). Brillouin microscopy can map the Brillouin-derived longitudinal modulus of the whole lens, but imaging times are lengthy. OCE can provide quantitative measurements of viscoelasticity rapidly, but the limited scattering of the lens limits its in-depth measurements. By combining these two techniques, we show a strong correlation between the Brillouin modulus and OCE-measured Young’s modulus in the lens, enabling depth-wise mapping of the Young’s modulus. The correlation coefficient between the two measurements was R=0.89. Using this correlation, the elasticity of the anterior lens was 2.72±0.89 kPa, and the mean Young’s modulus of the nucleus was 12.92±2.75 kPa. Similarly, the elasticity of the posterior lens was 3.80±1.25 kPa. While both techniques can evaluate the stiffness of the biological tissues separately, our work demonstrates that combining these techniques could enable mapping of the Young’s modulus completely noninvasively in non-scattering tissues such as the crystalline lens.
In the United States, 20% of pregnant women are estimated to smoke, thus affecting 800,000 babies annually. Maternal nicotine exposure is known to have several detrimental effects on the developing fetus including intrauterine growth restriction, perinatal mortality and morbidity, placental abruption, and other childhood disorders. In humans, studies evaluating the association between maternal cigarette smoking during pregnancy and behavioral development in offsprings have shown negative influences of nicotine on brain development. Although several studies have documented lower birth weights, morphological and behavioral changes, not much has been done evaluating the acute changes in brain vasculature after prenatal exposure to nicotine. This work uses correlation mapping optical coherence angiography (cm-OCA), a functional extension of optical coherence tomography, to evaluate changes in murine fetal brain vasculature, in utero, minutes after maternal nicotine exposure. A rapid and significant decrease in vasculature was observed compared to the sham group.
Prenatal substance abuse is one of the main causes of birth defects. Depending upon the substance being abused and the period of gestation during which the abuse happens, the severity of the defect is determined. Although prenatal substance abuse during the first trimester is common, the prevalence of unplanned pregnancies in the United States have led to women continuing their substance abuse well into the second trimester. The second trimester is the peak period for fetal neurogenesis and angiogenesis. Hence, any exposure to teratogens during this period is known to hinder brain development. Several studies have documented changes in morphology and behavior due to exposure to teratogens during this period. However, not a lot is known about the changes in vasculature in the developing brain. In this study, we used angiographic optical coherence tomography, a functional extension of optical coherence tomography, to image acute vasculature changes in the fetal brain caused due to prenatal exposure to ethanol, nicotine, and synthetic cannabinoids (SCB). Results showed a significant decrease in vasculature in all three cases compared to their respective sham groups.
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