Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Disruption of Fuz in mouse embryos generates hypoplastic hindbrain development and reduced cranial nerve ganglia.

BACKGROUND: The brain and spinal cord formation is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Environmental or genetic interferences can impair neurulation, resulting in clinically significant birth defects known collectively as neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. RESULTS: We demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent reduction of ventral neuroepithelial stiffness in a notochord adjacent area at the level of the rhombomere 5. The formation of cranial and paravertebral ganglia is also impaired in these embryos. CONCLUSIONS: This study reveals that hypoplastic hindbrain development, identified by abnormal rhombomere morphology and persistent loss of ventral neuroepithelial stiffness, precedes exencephaly in Fuz ablated murine mutants, indicating that the gene Fuz has a critical function sustaining normal neural tube development and neuronal differentiation.

Reverberant optical coherence elastography using multifocal acoustic radiation force.

In this study, we introduce a multifocal acoustic radiation force source that combines an ultrasound transducer and a 3D-printed acoustic lens for application in reverberant optical coherence elastography (Rev-OCE). An array of plano-concave acoustic lenses, each with an 11.8 mm aperture diameter, were used to spatially distribute the acoustic energy generated by a 1 MHz planar ultrasound transducer, producing multiple focal spots on a target plane. These focal spots generate reverberant shear wave fields detected by the optical coherence tomography (OCT) system. The effectiveness of the multifocal Rev-OCE system in probing mechanical properties with high resolution is demonstrated in layered gelatin phantoms.

Multimodal imaging system combining optical coherence tomography and Brillouin microscopy for neural tube imaging.

To understand the dynamics of tissue stiffness during neural tube formation and closure in a murine model, we have developed a multimodal, coaligned imaging system combining optical coherence tomography (OCT) and Brillouin microscopy. Brillouin microscopy can map the longitudinal modulus of tissue but cannot provide structural images. Thus, it is limited for imaging dynamic processes such as neural tube formation and closure. To overcome this limitation, we have combined Brillouin microscopy and OCT in one coaligned instrument. OCT provided depth-resolved structural imaging with a micrometer-scale spatial resolution to guide stiffness mapping by Brillouin modality. 2D structural and Brillouin frequency shift maps were acquired of mouse embryos at gestational day (GD) 8.5, 9.5, and 10.5 with the multimodal system. The results demonstrate the capability of the system to obtain structural and stiffness information simultaneously.

Prenatal alcohol exposure contributes to negative pregnancy outcomes by altering fetal vascular dynamics and the placental transcriptome.

BACKGROUND: Prenatal alcohol exposure (PAE) has been shown to alter fetal blood flow in utero and is also associated with placental insufficiency and intrauterine growth restriction (IUGR), suggesting an underlying connection between perturbed circulation and pregnancy outcomes. METHODS: Timed-pregnant C57/BL6NHsd mice, bred in-house, were exposed by gavage on gestational day 10 (GD10) to ethanol (3 g/kg) or purified water, as a control. Pulse-wave Doppler ultrasound measurements for umbilical arteries and ascending aorta were obtained post-gavage (GD12, GD14, GD18) on 2 fetuses/litter. RNA from the non-decidual (labyrinthine and junctional zone) portion of placentas was isolated and processed for RNA-seq and subsequent bioinformatic analyses, and the association between transcriptomic changes and fetal phenotypes assessed. RESULTS: Exposure to ethanol in pregnant mice on GD10 attenuates umbilical cord blood flow transiently during gestation, and is associated with indices of IUGR, specifically decreased fetal weight and morphometric indices of cranial growth. Moreover, RNA-seq of the fetal portion of the placenta demonstrated that this single exposure has lasting transcriptomic changes, including upregulation of Tet3, which is associated with spontaneous abortion. Weighted gene co-expression network analysis (WGCNA) identified erythrocyte differentiation and homeostasis as important pathways associated with improved umbilical cord blood flow as gestation progresses. WGCNA also identified sensory perception of chemical stimulus/odorant and receptor activity as important pathways associated with cranial growth. CONCLUSION: Our data suggest that PAE perturbs the expression of placental genes relevant for placental hematopoiesis and environmental sensing, resulting in transient impairment of umbilical cord blood flow and, subsequently, IUGR.

Introduction to optical coherence elastography: tutorial.

Optical coherence elastography (OCE) has seen rapid growth since its introduction in 1998. The past few decades have seen tremendous advancements in the development of OCE technology and a wide range of applications, including the first clinical applications. This tutorial introduces the basics of solid mechanics, which form the foundation of all elastography methods. We then describe how OCE measurements of tissue motion can be used to quantify tissue biomechanical parameters. We also detail various types of excitation methods, imaging systems, acquisition schemes, and data processing algorithms and how various parameters associated with each step of OCE imaging can affect the final quantitation of biomechanical properties. Finally, we discuss the future of OCE, its potential, and the next steps required for OCE to become an established medical imaging technology.

Ultra-fast dynamic line-field optical coherence elastography.

In this work, we present an ultra-fast line-field optical coherence elastography system (LF-OCE) with an 11.5 MHz equivalent A-line rate. The system was composed of a line-field spectral domain optical coherence tomography system based on a supercontinuum light source, Michelson-type interferometer, and a high-speed 2D spectrometer. The system performed ultra-fast imaging of elastic waves in tissue-mimicking phantoms of various elasticities. The results corroborated well with mechanical testing. Following validation, LF-OCE measurements were made in in situ and in in vivo rabbit corneas under various conditions. The results show the capability of the system to rapidly image elastic waves in tissues.

Multimodal high-resolution embryonic imaging with light sheet fluorescence microscopy and optical coherence tomography.

A high-resolution imaging system combining optical coherence tomography (OCT) and light sheet fluorescence microscopy (LSFM) was developed. LSFM confined the excitation to only the focal plane, removing the out of plane fluorescence. This enabled imaging a murine embryo with higher speed and specificity than traditional fluorescence microscopy. OCT gives information about the structure of the embryo from the same plane illuminated by LSFM. The co-planar OCT and LSFM instrument was capable of performing co-registered functional and structural imaging of mouse embryos simultaneously.

Micro Air-Pulse Spatial Deformation Spreading Characterizes Degree of Anisotropy in Tissues.

In optical coherence elastography (OCE), air-pulse stimulation has been widely used to produce propagation of mechanical waves for elastic characterization of tissues. In this paper, we propose the use of spatial deformation spreading (SDS) on the surface of samples produced by air-pulse stimulation for the OCE of transverse isotropic tissues. Experiments in isotropic tissue-mimicking phantoms and anisotropic chicken tibialis muscle were conducted using a spectral-domain optical coherence tomography system synchronized with a confocal air-pulse stimulation. SDS measurements were compared with wave speeds values calculated at different propagation angles. We found an approximately linear relationship between shear wave speed and SDS in isotropic phantoms, which was confirmed with predictions made by the numerical integration of a wave propagation model. Experimental measurements in chicken muscle show a good agreement between SDS and surface wave speed taken along and across the axis of symmetry of the tissues, also called degree of anisotropy. In summary, these results demonstrated the capabilities of SDS produced by the air-pulse technique in measuring the shear elastic anisotropy of transverse isotropic tissues.

In Vivo Human Corneal Shear-wave Optical Coherence Elastography.

SIGNIFICANCE: A novel imaging technology, dynamic optical coherence elastography (OCE), was adapted for clinical noninvasive measurements of corneal biomechanics. PURPOSE: Determining corneal biomechanical properties is a long-standing challenge. Elasticity imaging methods have recently been developed and applied for clinical evaluation of soft tissues in cancer detection, atherosclerotic plaque evaluation, surgical guidance, and more. Here, we describe the use of dynamic OCE to characterize mechanical wave propagation in the human cornea in vivo, thus providing a method for clinical determination of corneal biomechanical properties. METHODS: High-resolution phase-sensitive optical coherence tomography imaging was combined with microliter air-pulse tissue stimulation to perform dynamic elasticity measurements in 18 eyes of nine participants. Low-pressure (0.1 mmHg), spatiotemporally discreet (150 µm, 800 µs) tissue stimulation produced submicron-scale tissue deformations that were measured at multiple positions over a 1-mm2 area. Surface wave velocity was measured and used to determine tissue stiffness. Elastic wave propagation velocity was measured and evaluated as a function of IOP and central corneal thickness. RESULTS: Submicron corneal surface displacement amplitude (range, 0.005 to 0.5 µm) responses were measured with high sensitivity (0.24 nm). Corneal elastic wave velocity ranged from 2.4 to 4.2 m/s (mean, 3.5; 95% confidence interval, 3.2 to 3.8 m/s) and was correlated with central corneal thickness (r = 0.64, P < .001) and IOP (r = 0.52, P = .02). CONCLUSIONS: Phase-sensitive optical coherence tomography imaging combined with microliter air-pulse mechanical tissue stimulation has sufficient detection sensitivity to observe submicron elastic wave propagation in corneal tissue. These measurements enable in vivo corneal stiffness determinations that will be further studied for use with disease detection and for monitoring clinical interventions.

Confocal air-coupled ultrasonic optical coherence elastography probe for quantitative biomechanics.

We present an air-coupled ultrasonic radiation force probe co-focused with a phase-sensitive optical coherence tomography (OCT) system for quantitative wave-based elastography. A custom-made 1 MHz spherically focused piezoelectric transducer with a concentric 10 mm wide circular opening allowed for confocal micro-excitation of waves and phase-sensitive OCT imaging. Phantom studies demonstrated the capabilities of this probe to produce quasi-harmonic excitation up to 4 kHz for generation of elastic waves. Experimental results in ocular tissues showed highly detailed 2D and 3D elasticity mapping using this approach with great potential for clinical translation.

Longitudinal elastic wave imaging using nanobomb optical coherence elastography: erratum.

We present an erratum to correct an inadvertent error made during the calculations of the in-focus fluence of pulsed laser used to excite nanoparticles [Opt. Lett.44, 3162 (2019)OPLEDP0146-959210.1364/OL.44.003162] and to update the conclusion regarding laser safety limits achieved with this type of excitation.

Longitudinal elastic wave imaging using nanobomb optical coherence elastography.

Wave-based optical coherence elastography (OCE) is a rapidly emerging technique for elasticity assessment of tissues having high displacement sensitivity and simple implementation. However, most current noncontact wave excitation techniques are unable to target a specific tissue site in 3D and rely on transversal scanning of the imaging beam. Here, we demonstrate that dye-loaded perfluorocarbon nanoparticles (nanobombs) excited by a pulsed laser can produce localized axially propagating longitudinal shear waves while adhering to the laser safety limit. A phase-correction method was developed and implemented to perform sensitive nanobomb elastography using a ∼1.5 MHz Fourier domain mode-locking laser. The nanobomb activation was also monitored by detecting photoacoustic signals. The highly localized elastic waves detected by the nanobomb OCE suggest the possibility of high-resolution 3D elastographic imaging.

Can We Improve Vaginal Tissue Healing Using Customized Devices: 3D Printing and Biomechanical Changes in Vaginal Tissue.

BACKGROUND: Determining biomechanical changes in vaginal tissue with tissue stretch is critical for understanding the role of mechanotransduction on vaginal tissue healing. Noncontact dynamic optical coherence elastography (OCE) can quantify biomechanical changes in vaginal tissues noninvasively. Improved vaginal tissue healing will reduce postoperative complications from vaginal surgery. AIMS: (1) To complete dimensional assessments (DAs) of the vaginal tract. (2) To elucidate biomechanical properties (BMP) of porcine vaginal tissues (PVT). (3) Compare BMPs of piglet and adult PVTs after placement of customized vaginal dilators (VD) by OCE and uniaxial mechanical testing (MT). METHODS: Pilot study using adult nulliparous pig and piglet PVTs (n = 20 each). DA of PVTs was performed using silicone molding. 3D-printed VDs were used to achieve different Relative Diameter Change (RDC) of the PVTs (no dilatation, and -50%, 0%, 50% RDC). Elastographic testing using OCE and MT. RESULTS: Using OCE, no significant differences (SD) were noted between adult and piglet PVT (p = 0.74) or by stretch direction (p = 0.300). SD was noted with increasing RDC (p = 0.023). Using MT, there were SD in tissue stiffness between adult and piglet PVT (p = 0.048), but no SD as a function of RDC (p = 0.750) or stretch direction (p = 0.592). CONCLUSIONS: This study quantified biomechanical changes in PVT with customized stretching by 3D printed VD using both OCE and MT. This work has implications for the mechanotransduction of vaginal wound healing and noninvasive assessment of vaginal diseases.

Nanobomb optical coherence elastography.

Wave-based optical elastography is rapidly emerging as a powerful technique for quantifying tissue biomechanical properties due to its noninvasive nature and high displacement sensitivity. However, current approaches are limited in their ability to produce high-frequency waves and highly localized mechanical stress. In this Letter, we demonstrate that the rapid liquid-to-gas phase transition of dye-loaded perfluorocarbon nanodroplets ("nanobombs") initiated by a pulsed laser can produce highly localized, high-frequency, and broadband elastic waves. The waves were detected by an ultra-fast line-field low-coherence holography system. For comparison, we also excited waves using a focused micro-air-pulse. Results from tissue-mimicking phantoms showed that the nanobombs produced elastic waves with frequencies up to ∼9 kHz, which was much greater than the ∼2 kHz waves excited by the air-pulse. Consequently, the nanobombs enabled more accurate quantification of sample viscoelasticity. Combined with their potential for functionalization, the nanobombs show promise for accurate and highly specific noncontact all-optical elastography.

Effects of Thickness on Corneal Biomechanical Properties Using Optical Coherence Elastography.

SIGNIFICANCE: Measured corneal biomechanical properties are driven by intraocular pressure, tissue thickness, and inherent material properties. We demonstrate tissue thickness as an important factor in the measurement of corneal biomechanics that can confound short-term effects due to UV riboflavin cross-linking (CXL) treatment. PURPOSE: We isolate the effects of tissue thickness on the measured corneal biomechanical properties using optical coherence elastography by experimentally altering the tissue hydration state and stiffness. METHODS: Dynamic optical coherence elastography was performed using phase-sensitive optical coherence tomography imaging to quantify the tissue deformation dynamics resulting from a spatially discrete, low-force air pulse (150-µm spot size; 0.8-millisecond duration; <10 Pa [<0.08 mmHg]). The time-dependent surface deformation is characterized by a viscoelastic tissue recovery response, quantified by an exponential decay constant-relaxation rate. Ex vivo rabbit globes (n = 10) with fixed intraocular pressure (15 mmHg) were topically instilled every 5 minutes with 0.9% saline for 60 minutes and 20% dextran for another 60 minutes. Measurements were made after every 20 minutes to determine the central corneal thickness (CCT) and the relaxation rates. Cross-linking treatment was performed on another 13 eyes, applying isotonic riboflavin (n = 6) and hypertonic riboflavin (n = 7) every 5 minutes for 30 minutes, followed by UV irradiation (365 nm, 3 mW/cm) for 30 minutes while instilling riboflavin. Central corneal thickness and relaxation rates were obtained before and after CXL treatment. RESULTS: Corneal thickness was positively correlated (R = 0.9) with relaxation rates. In the CXL-treated eyes, isotonic riboflavin did not affect CCT and showed a significant increase in relaxation rates (+10%; P = .01) from 2.29 ms to 2.53 ms. Hypertonic riboflavin showed a significant CCT decrease (-31%; P = .01) from 618 µm to 429 µm but showed little change in relaxation rates after CXL treatment. CONCLUSIONS: Corneal thickness and stiffness are correlated positively. A higher relaxation rate implied stiffer material properties after isotonic CXL treatment. Hypertonic CXL treatment results in a stiffness decrease that offsets the stiffness increase with CXL treatment.

Noncontact Elastic Wave Imaging Optical Coherence Elastography for Evaluating Changes in Corneal Elasticity Due to Crosslinking.

The mechanical properties of tissues can provide valuable information about tissue integrity and health and can assist in detecting and monitoring the progression of diseases such as keratoconus. Optical coherence elastography (OCE) is a rapidly emerging technique, which can assess localized mechanical contrast in tissues with micrometer spatial resolution. In this work we present a noncontact method of optical coherence elastography to evaluate the changes in the mechanical properties of the cornea after UV-induced collagen cross-linking. A focused air-pulse induced a low amplitude (µm scale) elastic wave, which then propagated radially and was imaged in three dimensions by a phase-stabilized swept source optical coherence tomography (PhS-SSOCT) system. The elastic wave velocity was translated to Young's modulus in agar phantoms of various concentrations. Additionally, the speed of the elastic wave significantly changed in porcine cornea before and after UV-induced corneal collagen cross-linking (CXL). Moreover, different layers of the cornea, such as the anterior stroma, posterior stroma, and inner region, could be discerned from the phase velocities of the elastic wave. Therefore, because of noncontact excitation and imaging, this method may be useful for in vivo detection of ocular diseases such as keratoconus and evaluation of therapeutic interventions such as CXL.

Phase-sensitive optical coherence elastography at 1.5 million A-Lines per second.

Shear-wave imaging optical coherence elastography (SWI-OCE) is an emerging method for 3D quantitative assessment of tissue local mechanical properties based on imaging and analysis of elastic wave propagation. Current methods for SWI-OCE involve multiple temporal optical coherence tomography scans (M-mode) at different spatial locations across tissue surface (B- and C-modes). This requires an excitation for each measurement position leading to clinically unacceptable long acquisition times up to tens of minutes. In this Letter, we demonstrate, for the first time, noncontact true kilohertz frame-rate OCE by combining a Fourier domain mode-locked swept source laser with an A-scan rate of ∼1.5 MHz and a focused air-pulse as an elastic wave excitation source. The propagation of the elastic wave in the sample was imaged at a frame rate of ∼7.3 kHz. Therefore, to quantify the elastic wave propagation velocity in a single direction, only a single excitation was needed. This method was validated by quantifying the elasticity of tissue-mimicking agar phantoms as well as of a porcine cornea ex vivo at different intraocular pressures. The results demonstrate that this method can reduce the acquisition time of an elastogram to milliseconds.

Direct four-dimensional structural and functional imaging of cardiovascular dynamics in mouse embryos with 1.5 MHz optical coherence tomography.

High-resolution three-dimensional (3D) imaging of cardiovascular dynamics in mouse embryos is greatly desired to study mammalian congenital cardiac defects. Here, we demonstrate direct four-dimensional (4D) imaging of the cardiovascular structure and function in live mouse embryos at a ∼43 Hz volume rate using an optical coherence tomography (OCT) system with a ∼1.5 MHz Fourier domain mode-locking swept laser source. Combining ultrafast OCT imaging with live mouse embryo culture protocols, 3D volumes of the embryo are directly and continuously acquired over time for a cardiodynamics analysis without the application of any synchronization algorithms. We present the time-resolved measurements of the heart wall motion based on the 4D structural data, report 4D speckle variance and Doppler imaging of the vascular system, and quantify spatially resolved blood flow velocity over time. These results indicate that the ultra-high-speed 4D imaging approach could be a useful tool for efficient cardiovascular phenotyping of mouse embryos.

Shear wave imaging optical coherence tomography (SWI-OCT) for ocular tissue biomechanics.

We report on a noncontact low-coherence optical phase-based imaging method, termed shear wave imaging optical coherence tomography (SWI-OCT), which enables 2D depth-resolved visualization of the low-amplitude elastic wave propagation in tissue with ultrahigh frame rate. SWI-OCT is based on 1D transverse scanning of the M-mode OCT imaging that is precisely synchronized with a low-pressure short-duration air-puff loading system. This approach of scanning and data recording allows visualization of the induced tissue deformation at high frame rate. The applied phase-resolved interferometric technique, with sensitivity on the nanometer scale, makes the low-amplitude tissue displacement detectable. For the demonstration of this method, and to study its application for tissue biomechanics, we performed pilot experiments on agar phantoms and ex vivo rabbit corneas. Samples with different elastic properties can be differentiated based on the velocity of the elastic wave propagation that is directly visualized with a 25 kHz frame rate. Our results indicate that SWI-OCT has the potential to be further developed as a major technique for depth-resolved high-resolution tissue elastography in vivo.