A feasibility study of OCT for anatomical and vascular phenotyping of mouse embryo

The embryo phenotyping of genetic murine model is invaluable when investigating functions of genes underlying embryonic development and birth defect. Although traditional imaging technologies such as ultrasound are very useful for evaluating phenotype of murine embryos, the use of advanced techniques for phenotyping is desirable to obtain more information from genetic research. This letter tests the feasibility of optical coherence tomography (OCT) as a high‐throughput phenotyping tool for murine embryos. Three‐dimensional OCT imaging is performed for live and cleared mouse embryos in the late developmental stage (embryonic day 17.5). By using a dynamic focusing method and OCT angiography (OCTA) approach, our OCT imaging of the embryo exhibits rapid and clean visualization of organ structures deeper than 5 mm and complex microvasculature of perfused blood vessels in the murine embryonic body. This demonstration suggests that OCT imaging can be useful for comprehensively assessing embryo anatomy and angiography of genetically engineered mice.     

In‐vivo hemodynamic imaging of acute prenatal ethanol exposure in fetal brain by photoacoustic tomography

Prenatal ethanol exposure (PEE) can lead to structural and functional abnormalities in fetal brain. Although neural developmental deficits due to PEE have been recognized, the immediate effects of PEE on fetal brain vasculature and hemodynamics remain poorly understood. One of the major obstacles that preclude the rapid advancement of studies on fetal vascular dynamics is the limitation of the imaging techniques. Thus, a technique for noninvasive in‐vivo imaging of fetal vasculature and hemodynamics is desirable. In this study, we explored the dynamic changes of the vessel dimeter, density and oxygen saturation in fetal brain after acute maternal ethanol exposure in the second‐trimester equivalent murine model using a real‐time photoacoustic tomography system we developed for imaging embryo of small animals. The results indicate a significant decrease in fetal brain vessel diameter, perfusion and oxygen saturation. This work demonstrated that PAT can provide high‐resolution noninvasive imaging ability to monitor fetal vascular dynamics.     

Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development

Noninvasive visualization of embryos at different development stages is crucial for the understanding of the basic developmental biology. It is therefore desirable to have an imaging tool capable of rapidly evaluating the effects of gene manipulation or genome editing in developing embryos for the studies of gene functions and genetic engineering. Here, we propose and demonstrate a novel use of optical coherence tomography (OCT) to noninvasively exam the embryonic development of the migratory locusts in real time with 3‐dimensional (3D) view capability. In particular, we obtain the sufficiently high spatial resolution tomographic 2D and 3D images of live locust embryos throughout their development processes. We show that not only we are able to noninvasively observe all previously known forms of blastokinesis as an embryo develops, such as anatrepsis, katatrepsis, revolution, rotation and diapauses, and determine their precise occurring time or duration, but also discover an unreported rotation form we named “twist.” In addition, with the OCT images we determined the exact occurring time of diapauses of the locusts from Tibetan plateau for the first time. Finally, we demonstrate that OCT systems can be used to rapidly capture the development defects of genetically modified embryos in which certain genes essential for embryonic development were suppressed by RNA interference. Our work shows that OCT is an enabling imaging tool with sufficient spatial resolution for the rapid evaluation of embryonic variations of small animals.     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Enhanced spatial resolution for snapshot hyperspectral imaging of blood perfusion and melanin information within human tissue

We report a reconstruction method to achieve high spatial resolution for hyperspectral imaging of chromophore features in skin in vivo. The method utilizes an established structure‐adaptive normalized convolution algorithm to reconstruct high spatial resolution of hyperspectral images from snapshot low‐resolution hyperspectral image sequences captured by a snapshot spectral camera. The reconstructed images at chromophore‐sensitive wavebands are used to map the skin features of interest. We demonstrate the method experimentally by mapping the blood perfusion and melanin features (moles) on the facial skin. The method relaxes the constrains of the relatively low spatial resolution in the snapshot hyperspectral camera, making it more usable in imaging applications.     

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

利用归一化互相关算法去除吸收强度涨落调制血管造影图像模糊

吸收强度涨落调制成像（AIFM）方法是基于血红细胞和背景组织对低相干光照明的吸收差异,通过在频域分离动态的血红细胞信号和静态的背景信号,实现对近透明活体生物样本全场无标记的光学血管造影成像. 但此成像方法需采集较长的原始图像序列,系统漂移或生物抖动会造成图像模糊,难以实现对某些特定区域的血管造影成像. 本文提出一种结合AIFM成像和归一化互相关算法的新方法来提升血管造影图像的质量：原始的图像序列被分成若干短时序列,每个短时序列先利用AIFM成像算法重构得到全场的血管造影图像;再利用归一化的互相关算法将所有的短时重构图像与第一帧重构图像相匹配,并融合得到最终的血管造影片. 我们以活体鸡蛋胚胎为样品,通过实验验证了利用短时归一化互相关AIFM成像方法,能够消除鸡胚胎心跳引起的图像模糊,从而获得高分辨率和信噪比的心血管造影片,对研究活体动物心脑血管疾病具有重要应用价值.     

Photoacoustic imaging of the human placental vasculature

Minimally invasive fetal interventions require accurate imaging from inside the uterine cavity. Twin‐to‐twin transfusion syndrome (TTTS), a condition considered in this study, occurs from abnormal vascular anastomoses in the placenta that allow blood to flow unevenly between the fetuses. Currently, TTTS is treated fetoscopically by identifying the anastomosing vessels, and then performing laser photocoagulation. However, white light fetoscopy provides limited visibility of placental vasculature, which can lead to missed anastomoses or incomplete photocoagulation. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, two PA systems were used to visualize chorionic (fetal) superficial and subsurface vasculature in human placentas. The first system comprised an optical parametric oscillator for PA excitation and a 2D Fabry‐Pérot cavity ultrasound sensor; the second, light emitting diode arrays and a 1D clinical linear‐array ultrasound imaging probe. Volumetric photoacoustic images were acquired from ex vivo normal term and TTTS‐treated placentas. It was shown that superficial and subsurface branching blood vessels could be visualized to depths of approximately 7 mm, and that ablated tissue yielded negative image contrast. This study demonstrated the strong potential of PA imaging to guide minimally invasive fetal therapies.      

Noninvasive Imaging of Ethanol‐Induced Developmental Defects in Zebrafish Embryos Using Optical Coherence Tomography

In this article, we report the use of optical coherence tomography for noninvasive cross‐sectional real‐time imaging of ethanol‐induced developmental defects in zebrafish embryos larvae. For ethanol concentration of over 300 mM, developmental defects of eye (shrinkage and retinal abnormalities), malformation of the notochord and ataxia arising due to the toxic effects of ethanol were observed in OCT images from 3 days post fertilization onwards. The results suggest that OCT could be a valuable tool for noninvasive assessment of birth defects in small animal systems.     

Staging mouse preimplantation development in vivo using optical coherence microscopy

In mammals, preimplantation development primarily occurs in the oviduct (or fallopian tube) where fertilized oocytes migrate through, develop and divide as they prepare for implantation in the uterus. Studies of preimplantation development currently rely on ex vivo experiments with the embryos cultured outside of the oviduct, neglecting the native environment for embryonic growth. This prevents the understanding of the natural process of preimplantation development and the roles of the oviduct in early embryonic health. Here, we report an in vivo optical imaging approach enabling high‐resolution visualizations of developing embryos in the mouse oviduct. By combining optical coherence microscopy (OCM) and a dorsal imaging window, the subcellular structures and morphologies of unfertilized oocytes, zygotes and preimplantation embryos can be well resolved in vivo, allowing for the staging of development. We present the results together with bright‐field microscopy images to show the comparable imaging quality. As the mouse is a well‐established model with a variety of genetic engineering strategies available, the in vivo imaging approach opens great opportunities to investigate how the oviduct and early embryos interact to prepare for successful implantation. This knowledge could have beneficial impact on understanding infertility and improving in vitro fertilization. OCM through a dorsal imaging window enables high‐resolution imaging and staging of mouse preimplantation embryos in vivo in the oviduct.      

Detecting hemodynamic changes in the foot vessels of diabetic patients by photoacoustic tomography

Limb perfusion monitoring is critical for diabetes mellitus (DM) patients as they are vulnerable to vascular complications due to prolonged hyperglycemia. However, current clinical approaches are ineffective in vascular imaging and in assessing vascular function in lower limbs. In this work, a concave ultrasound transducer array‐based photoacoustic tomography (PAT) system was used to image the foot dorsal section of a subject, and a total of seven DM patients and seven healthy volunteers were enrolled in this study. Hemodynamic changes in foot vessels during vascular occlusion as well as oxygen saturation (SO₂) in rest were analyzed for both groups. The results obtained showed that DM patients have a unique peripheral hemodynamic response to occlusion and a lower level SO₂, compared to that for healthy subjects. This suggests that PAT has the potential to detect vascular dysfunction in DM patients and to measure the effect of treatment.     

Fan‐shaped scanning approach for miniaturized photoacoustic tomography

We describe a novel scanning approach for miniaturized photoacoustic tomography (PAT), based on fan‐shaped scanning of a single transducer at one or two discrete positions. This approach is tested and evaluated using several phantom and animal experiments. The results obtained show that this new scanning approach provides high image quality in the configuration of miniaturized handheld or endoscopic PAT with improved effective field of view and penetration depth.     

Noninvasive photoacoustic and fluorescent tracking of optical dye labeled T cellular activities of diseased sites at new depth

The migration of immune cells is crucial to the immune response. Visualization of these processes has previously been limited because of the imaging depth. We developed a deep‐penetrating, sensitive and high‐resolution method to use fast photoacoustic tomography (PAT) to image the dynamic changes of T cells in lymph node and diseases at new depth (up to 9.5 mm). T cells labeled with NIR‐797‐isothiocyanate, an excellent near‐infrared photoacoustic and fluorescent agent, were intravenously injected to the mice. We used fluorescence imaging to determine the location of T cells roughly and photoacoustic imaging is used to observe T‐cell responses in diseased sites deeply and carefully. The dynamic changes of T cells in lymph node, acute disease (bacterial infection) and chronic disease (tumor) were observed noninvasively by photoacoustic and fluorescence imaging at different time points. T cells accumulated gradually and reached a maximum at 4 hours and declined afterwards in lymph node and bacterial infection site. At tumor model, T cells immigrated to the tumor with a maximum at 12 hours. Our study can not only provide a new observing method for immune activities tracking, but also enable continuous monitoring for therapeutic interventions.      

Impacts of the murine skull on high‐frequency transcranial photoacoustic brain imaging

Non‐invasive photoacoustic tomography (PAT) of mouse brains with intact skulls has been a challenge due to the skull's strong acoustic attenuation, aberration, and reverberation, especially in the high‐frequency range (>15 MHz). In this paper, we systematically investigated the impacts of the murine skull on the photoacoustic wave propagation and on the PAT image reconstruction. We studied the photoacoustic acoustic wave aberration due to the acoustic impedance mismatch at the skull boundaries and the mode conversion between the longitudinal wave and shear wave. The wave's reverberation within the skull was investigated for both longitudinal and shear modes. In the inverse process, we reconstructed the transcranial photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) images of a point target enclosed by the mouse skull, showing the skull's different impacts on both modalities. Finally, we experimentally validated the simulations by imaging an in vitro mouse skull phantom using representative transcranial PAM and PACT systems. The experimental results agreed well with the simulations and confirmed the accuracy of our forward and inverse models. We expect that our results will provide better understanding of the impacts of the murine skull on transcranial photoacoustic brain imaging and pave the ways for future technical improvements.      

In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell–specific, fluorescent reporter mouse skin with induced ischemia‐reperfusion (I‐R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual‐wavelength micro electro mechanical system (MEMS) scanning–based optical resolution photoacoustic microscopy (OR‐PAM) system for continuous label‐free functional imaging of vascular reperfusion in an IR mouse model. This MEMS‐OR‐PAM system provides fast scanning speed for concurrent dual‐wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO₂) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS‐OR‐PAM system provides novel perspectives to understand the I‐R injuries. It solves the problem of dynamic label‐free functional monitoring of the vascular reperfusion at high spatial resolution.      

In vivo blind‐deconvolution photoacoustic ophthalmoscopy with total variation regularization

Photoacoustic ophthalmoscopy (PAOM) is capable of noninvasively imaging anatomic and functional information of the retina in living rodents. However, the strong ocular aberration in rodent eyes and limited ultrasonic detection sensitivity affect PAOM's spatial resolution and signal‐to‐noise ratio (SNR) in in vivo eyes. In this work, we report a computational approach to combine blind deconvolution (BD) algorithm with a regularizing constraint based on total variation (BDTV) for PAOM imaging restoration. We tested the algorithm in retinal and choroidal microvascular images in albino rat eyes. The algorithm improved PAOM's lateral resolution by around 2‐fold. Moreover, it enabled the improvement in imaging SNR for both major vessels and capillaries, and realized the well‐preserved blood vessels' edges simultaneously, which surpasses conventional Richardson‐Lucy BD algorithm. The reported results indicate that the BDTV algorithm potentially facilitate PAOM in extracting retinal pathophysiological information by enhancing in vivo imaging quality without physically modifying PAOM's optical configuration.