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.

Noninvasive Tracking of Embryonic Cardiac Dynamics and Development with Volumetric Optoacoustic Spectroscopy.

Noninvasive monitoring of cardiac development can potentially prevent cardiac anomalies in adulthood. Mouse models provide unique opportunities to study cardiac development and disease in mammals. However, high-resolution noninvasive functional analyses of murine embryonic cardiac models are challenging because of the small size and fast volumetric motion of the embryonic heart, which is deeply embedded inside the uterus. In this study, a real time volumetric optoacoustic spectroscopy (VOS) platform for whole-heart visualization with high spatial (100 µm) and temporal (10 ms) resolutions is developed. Embryonic heart development on gestational days (GDs) 14.5-17.5 and quantify cardiac dynamics using time-lapse-4D image data of the heart is followed. Additionally, spectroscopic recordings enable the quantification of the blood oxygenation status in heart chambers in a label-free and noninvasive manner. This technology introduces new possibilities for high-resolution quantification of embryonic heart function at different gestational stages in mammalian models, offering an invaluable noninvasive method for developmental biology.

Red light instruments for myopia exceed safety limits.

PURPOSE: Low-level red light (LLRL) therapy has recently emerged as a myopia treatment in children, with several studies reporting significant reduction in axial elongation and myopia progression. The goal of this study was to characterise the output and determine the thermal and photochemical maximum permissible exposure (MPE) of LLRL devices for myopia control. METHODS: Two LLRL devices, a Sky-n1201a and a Future Vision, were examined. Optical power measurements were made using an integrating sphere radiometer through a 7-mm diameter aperture, in accordance with ANSI Z136.1-2014, sections 3.2.3-3.2.4. Retinal spot sizes of the devices were obtained using a model eye and high-resolution beam profiler. Corneal irradiance, retinal irradiance and MPE were calculated for an eye positioned at the oculars of each device. RESULTS: Both devices were confirmed to be Class 1 laser products. Findings showed that the Sky-n1201a delivers laser light as a point source with a 654-nm wavelength, 0.2 mW power (Ø 7 mm aperture, 10-cm distance), 1.17 mW/cm2 corneal irradiance and 7.2 W/cm2 retinal irradiance (Ø 2 mm pupil). The MPE for photochemical damage is 0.55-7.0 s for 2-7 mm pupils and for thermal damage is 0.41-10 s for 4.25-7 mm pupils. Future Vision delivers the laser as an extended source subtending 0.75 × 0.325°. It has a 652-nm wavelength, 0.06 mW power (Ø 7 mm aperture, 10 cm distance), 0.624 mW/cm2 corneal irradiance and 0.08 W/cm2 retinal irradiance (Ø 2 mm pupil). MPE for photochemical damage is 50-625 s for 2-7 mm pupils. DISCUSSION: For both of the LLRL devices evaluated here, 3 min of continuous viewing approached or surpassed the MPE, putting the retina at risk of photochemical and thermal damage. Clinicians should be cautious with the use of LLRL therapy for myopia in children until safety standards can be confirmed.

Nanobomb optical coherence elastography in multilayered phantoms.

Many tissues are composed of layered structures, and a better understanding of the changes in the layered tissue biomechanics can enable advanced guidance and monitoring of therapy. The advent of elastography using longitudinally propagating shear waves (LSWs) has created the prospect of a high-resolution assessment of depth-dependent tissue elasticity. Laser activation of liquid-to-gas phase transition of dye-loaded perfluorocarbon (PFC) nanodroplets (a.k.a., nanobombs) can produce highly localized LSWs. This study aims to leverage the potential of photoactivation of nanobombs to incudce LSWs with very high-frequency content in wave-based optical coherence elastography (OCE) to estimate the elasticity gradient with high resolution. In this work, we used multilayered tissue-mimicking phantoms to demonstrate that highly localized nanobomb (NB)-induced LSWs can discriminate depth-wise tissue elasticity gradients. The results show that the NB-induced LSWs rapidly change speed when transitioning between layers with different mechanical properties, resulting in an elasticity resolution of ∼65 µm. These results show promise for characterizing the elasticity of multilayer tissue with a fine resolution.

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.

A novel multipurpose device for guided knee motion and loading during dynamic magnetic resonance imaging.

INTRODUCTION: This work aimed to develop a novel multipurpose device for guided knee flexion-extension, both passively using a motorized pneumatic system and actively (muscle-driven) with the joint unloaded or loaded during dynamic MRI. Secondary objectives were to characterize the participant experience during device use, and present preliminary dynamic MRI data to demonstrate the different device capabilities. MATERIAL AND METHODS: Self-reported outcomes were used to characterize the pain, physical exertion and discomfort levels experienced by 10 healthy male participants during four different active knee motion and loading protocols using the novel device. Knee angular data were recorded during the protocols to determine the maximum knee range of motion achievable. Dynamic MRI was acquired for three healthy volunteers during passive, unloaded knee motion using 2D Cartesian TSE, 2D radial GRE and 3D UTE sequences; and during active, unloaded and loaded knee motion using 2D radial GRE imaging. Because of the different MRI sequences used, spatial resolution was inherently lower for active knee motion than for passive motion acquisitions. RESULTS: Depending on the protocol, some participants reported slight pain, mild discomfort and varying levels of physical exertion. On average, participants achieved ∼40° of knee flexion; loaded conditions can create knee moments up to 27Nm. High quality imaging data were obtained during different motion and loading conditions. Dynamic 3D data allowed to retrospectively extract arbitrarily oriented slices. CONCLUSION: A novel multipurpose device for guided, physiologically relevant knee motion and loading during dynamic MRI was developed. Device use was well tolerated and suitable for acquiring high quality images during different motion and loading conditions. Different bone positions between loaded and unloaded conditions were likely due to out-of-plane motion, particularly because image registration was not performed. Ultimately, this device could be used to advance our understanding of physiological and pathological joint mechanics.

Longitudinal In Vivo Changes in Radial Peripapillary Capillaries and Optic Nerve Head Structure in Non-Human Primates With Early Experimental Glaucoma.

Purpose: There is conflicting evidence regarding whether a loss of radial peripapillary capillaries (RPCs) precedes neuronal loss in glaucoma. We examined the time course of in vivo changes in RPCs, optic nerve head (ONH) structure, and retinal nerve fiber layer thickness (RNFLT) in experimental glaucoma (EG). Methods: Spectral domain optical coherence tomography images were acquired before and approximately every two weeks after inducing unilateral EG in nine rhesus monkeys to quantify mean anterior lamina cribrosa surface depth (ALCSD), minimum rim width (MRW), and RNFLT. Perfused RPC density was measured from adaptive optics scanning laser ophthalmoscope images acquired on the temporal half of the ONH. The time of first significant change was quantified as when values fell and remained outside of the 95% confidence interval established from control eyes. Results: Mean ALCSD and/or MRW were the first parameters to change in eight EG eyes. RPC density changed first in the ninth. At their first points of change, mean ALCSD posteriorly deformed by 100.2 ± 101.2 µm, MRW thinned by 82.3 ± 65.9 µm, RNFLT decreased by 25 ± 14 µm, and RPC density decreased by 4.5 ± 2.1%. RPC density decreased before RNFL thinning in 5 EG eyes. RNFLT decreased before RPC density decreased in two EG eyes, whereas two EG eyes had simultaneous changes. Conclusions: In most EG eyes, RPC density decreased before (or simultaneous with) a change in RNFLT, suggesting that vascular factors may play a role in axonal loss in some eyes in early glaucoma.

Clinical applications of personalising the neural components of visual image quality metrics for individual eyes.

PURPOSE: Eyecare is evolving increasingly personalised corrections and increasingly personalised evaluations of corrections on-eye. This report describes individualising optical and neural components of the VSX (visual Strehl) metric and evaluates personalisation using two clinical applications. (1) Better understanding visual experience: While VSX tracks visual performance in typical eyes, non-individualised metrics underestimated visual performance in highly aberrated eyes - could this be understood by personalising metrics? (2) Metric-optimised objective spherocylindrical refractions in typical and atypical eyes have used neural weighting functions of typical eyes - will personalisation affect the outcome in clinical 0.25D steps? METHODS: Orientation-specific neural contrast sensitivity was measured in four typical myopic and astigmatic eyes and six eyes with keratoconus. Wavefront error was measured in all eyes while uncorrected and when the keratoconic eyes wore wavefront-guided scleral lenses. Total experiment duration was 24-28 h per subject. Two versions of VSX were calculated for each application: one weighted ocular optics with measured neural contrast sensitivity data from that eye, another weighted optics with a representative neural function of typical eyes. Wavefront-guided corrections were evaluated using the two metric values. Spherocylindrical corrections that optimised each metric were identified. RESULTS: Metric values for keratoconic eyes improved by a mean factor of 1.99 (~0.3 log units) when personalised. Applying this factor to a larger sample of eyes from a previous keratoconus study reconciled dissonances between the percentage of eyes reaching normative best-corrected metric levels and the percentages of eyes reaching normative levels of visual acuity and contrast sensitivity. Spherocylindrical corrections that optimised both versions of VSX were clinically equivalent (mean ± SD Euclidean dioptric difference 0.13 ± 0.18 D). CONCLUSIONS: Personalising visual image quality metrics is beneficial when actual metric values are used (evaluating ophthalmic corrections on-eye against norms) and when fine increments in visual quality are imparted (wavefront-guided corrections). However, partially individualised metrics appear adequate when metrics relatively rank spherocylindrical corrections in 0.25 D steps.

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.

Orientation-specific long-term neural adaptation of the visual system in keratoconus.

Eyes with the corneal ectasia keratoconus have performed better than expected (e.g. visual acuity) given their elevated levels of higher-order aberrations that cause rotationally asymmetric retinal blur. Adapted neural processing has been suggested as an explanation but has not been measured across multiple meridional orientations. Using a custom Maxwellian-view laser interferometer to bypass ocular optics, sinusoidal grating neural contrast sensitivity was measured in six eyes (three subjects) with keratoconus and four typical eyes (two subjects) at six spatial frequencies and eight orientations using a two-interval forced-choice paradigm. Total measurement duration was 24 to 28 hours per subject. Neural contrast sensitivity functions of typical eyes agreed with literature and generally showed the oblique effect on a linear-scale and rotational symmetry on a log-scale (rotational symmetry was quantified as the ratio of the minor and major radii of an ellipse fit to all orientations within each spatial frequency; typical eye mean 0.93, median 0.93; where a circle = 1). Mean sensitivities of eyes with keratoconus were 20% to 60% lower (at lower and higher spatial frequencies respectively) than typical eyes. Orientation-specific neural contrast sensitivity functions in keratoconus showed substantial rotational asymmetry (ellipse radii ratio: mean 0.84; median 0.86) and large meridional reductions. The visual image quality metric VSX was used with a permutation test to combine the asymmetric optical aberrations of the eyes with keratoconus and their measured asymmetric neural functions, which illustrated how the neural sensitivities generally mitigated the detrimental effects of the optics.

Automatic Segmentation of Retinal Capillaries in Adaptive Optics Scanning Laser Ophthalmoscope Perfusion Images Using a Convolutional Neural Network.

Purpose: Adaptive optics scanning laser ophthalmoscope (AOSLO) capillary perfusion images can possess large variations in contrast, intensity, and background signal, thereby limiting the use of global or adaptive thresholding techniques for automatic segmentation. We sought to develop an automated approach to segment perfused capillaries in AOSLO images. Methods: 12,979 image patches were extracted from manually segmented AOSLO montages from 14 eyes and used to train a convolutional neural network (CNN) that classified pixels as capillaries, large vessels, background, or image canvas. 1764 patches were extracted from AOSLO montages of four separate subjects, and were segmented manually by two raters (ground truth) and automatically by the CNN, an Otsu's approach, and a Frangi approach. A modified Dice coefficient was created to account for slight spatial differences between the same manually and CNN-segmented capillaries. Results: CNN capillary segmentation had an accuracy (0.94), a Dice coefficient (0.67), and a modified Dice coefficient (0.90) that were significantly higher than other automated approaches (P < 0.05). There were no significant differences in capillary density and mean segment length between manual ground-truth and CNN segmentations (P > 0.05). Conclusions: Close agreement between the CNN and manual segmentations enables robust and objective quantification of perfused capillary metrics. The developed CNN is time and computationally efficient, and distinguishes capillaries from areas containing diffuse background signal and larger underlying vessels. Translational Relevance: This automatic segmentation algorithm greatly increases the efficiency of quantifying AOSLO capillary perfusion images.

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.

Laser-induced elastic wave classification: thermoelastic versus ablative regimes for all-optical elastography applications.

SIGNIFICANCE: Shear wave optical coherence elastography is an emerging technique for characterizing tissue biomechanics that relies on the generation of elastic waves to obtain the mechanical contrast. Various techniques, such as contact, acoustic, and pneumatic methods, have been used to induce elastic waves. However, the lack of higher-frequency components within the elastic wave restricts their use in thin samples. The methods also require moving parts and/or tubing, which therefore limits the extent to which they can be miniaturized. AIM: To overcome these limitations, we propose an all-optical approach using photothermal excitation. Depending on the absorption coefficient of the sample and the laser pulse energy, elastic waves are generated either through a thermoelastic or an ablative process. Our study aimed to experimentally determine the boundary between the thermoelastic and the ablative regimes for safe all-optical elastography applications. APPROACH: Tissue-mimicking graphite-doped phantoms and chicken liver samples were used to investigate the boundary between thermoelastic and ablative regimes. A pulsed laser at 532 nm was used to induce elastic waves in the samples. Laser-induced elastic waves were detected using a line field low coherence holography instrument. The shape of the elastic wave amplitude was analyzed and used to determine the transition point between thermoelastic and ablative regimes. RESULTS: The transition from the thermoelastic to the ablative regime is accompanied by the nonlinear increase in surface wave amplitude as well as the transformation of the wave shape. Correlation between the absorption coefficient and the transition point energy was experimentally determined using graphite-doped phantoms and applied to biological samples ex vivo. CONCLUSIONS: Our study described a methodology for determining the boundary region between thermoelastic and ablative regimes of elastic wave generation. These can be used for the development of a safe method for completely noncontact, all-optical microscale assessment of tissue biomechanics using laser-induced elastic waves.

Translational optical coherence elastography for assessment of systemic sclerosis.

Systemic sclerosis (SSc-scleroderma) is an autoimmune disorder with high mortality rate that results in excessive accumulation of collagen in the skin and internal organs. Currently, the modified Rodnan Skin Score (mRSS) is the gold standard for evaluating the dermal thickening due to SSc. However, mRSS has noticeable inter- and intra-observer variabilities as quantified by the interclass correlation coefficient (ICC: 0.6-0.75). In this work, optical coherence elastography (OCE) combined with structural optical coherence tomography (OCT) image analysis was used to assess skin thickness in 12 SSc patients and healthy volunteers. Inter- (ICC: 0.62-0.99) and intra-observer (ICC > 0.90) assessment of OCT/OCE showed excellent reliability. Clinical assessments, including histologically assessed dermal thickness (DT), mRSS, and site-specific mRSS (SMRSS) were also performed for further validation. The OCE and OCT results from the forearm demonstrated the highest correlation (OCE: 0.78, OCT: 0.65) with SMRSS. Importantly, OCE and OCT had stronger correlations with the histological DT (OCT: r = .78 and OCE: r = .74) than SMRSS (r = .57), indicating the OCT/OCE could outperform semi-quantitative clinical assessments such as SMRSS. Overall, these results demonstrate that OCT/OCE could be useful for rapid, noninvasive and objective assessments of SSc onset and monitoring skin disease progression and treatment response.

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.

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.

Ultra-fast line-field low coherence holographic elastography using spatial phase shifting.

Optical coherence elastography (OCE) is an emerging technique for quantifying tissue biomechanical properties. Generally, OCE relies on point-by-point scanning. However, long acquisition times make point-by-point scanning unfeasible for clinical use. Here we demonstrate a noncontact single shot line-field low coherence holography system utilizing an automatic Hilbert transform analysis based on a spatial phase shifting technique. Spatio-temporal maps of elastic wave propagation were acquired with only one air-pulse excitation and used to quantify wave velocity and sample mechanical properties at a line rate of 200 kHz. Results obtained on phantoms were correlated with data from mechanical testing. Finally, the stiffness of porcine cornea at different intraocular pressures was also quantified in situ.