Quantitative optical imaging of cilia-driven fluid flow

描述（由申请人提供）：呼吸系统疾病是儿科发病和死亡的主要原因。对这些疾病的了解不完全，这是改善临床护理的障碍。因此，需要发现疾病的新机制。光学成像（例如光学相干断层扫描[OCT]）将使这些发现成为可能，因为传统成像（例如X射线、CT、MRI）不能可视化小于~1 mm的结构。微流体尺度纤毛驱动的流体流动将病原体和含有过敏原的粘液清除出肺部，但我们目前缺乏定量成像技术来表征其流动性能。此外，虽然传统上认为纤毛缺陷是罕见但严重疾病（例如原发性纤毛运动障碍）的特征，但从定量血流成像中提取的生物力学生物标志物的发展将使我们能够测试范式转移假设：目前诊断无法检测到的纤毛性能的中间缺陷是常见呼吸系统疾病（例如哮喘）临床严重程度的主要修饰符。因此，我们的研究有三个目的。首先，我们将开发高速、无余弦模糊度的多普勒OCT成像系统。纤毛驱动的流体流动本质上是三维的，并且不服从简化的几何假设，例如抛物线流动剖面。传统的多普勒成像具有余弦模糊性，其排除了三分量流速（v=vxi+vyj+vzk）的测量。我们将开发一种新型的OCT干涉仪，使三维，三组分流动成像，将使用爪蟾（蝌蚪）胚胎，纤毛生物学中的一个重要动物模型的纤毛皮肤证明。第二，我们将开发利用纤毛驱动的微流体技术的纤毛功能的定量成像分析。 混合从仿生纤毛的工作中得到启示，我们已经（a）证明了纤毛生物表面可以驱动微流体混合，（B）开发了一种新型的微流体芯片，该芯片使用纤毛生物表面作为微流体“组件”。“在这些结果的基础上，我们将证明我们的基于微流体混合的测定可以量化纤毛生理学的生物相关扰动，包括增加的流体粘度和改变的平面细胞极性。第三，我们将证明中间缺陷爪蟾和小鼠睫状体功能使用定量成像。我们将针对与爪蟾胚胎中纤毛表面的性能相关的两种不同类型的基因：纤毛分子马达和notch信号蛋白（notch信号控制胚胎皮肤上纤毛细胞的密度）。鉴于小儿呼吸道疾病小鼠模型的重要性，证明我们的光学方法可用于量化小鼠呼吸纤毛的性能至关重要。此外，这是将我们的诊断技术用于人类的重要一步。我们建议使用血流成像来量化因液体粘度增加（机械扰动以降低纤毛搏动频率）和细胞外ATP增加（药理学扰动以增加纤毛搏动频率）而改变的性能。

DESCRIPTION (provided by applicant): Respiratory diseases are major causes of pediatric morbidity and mortality. These diseases are incompletely understood, which is a barrier to improving clinical care. Therefore, new mechanisms of disease need to be discovered. Optical imaging (e.g. optical coherence tomography [OCT]) will enable these discoveries since traditional imaging (e.g. x-ray, CT, MRI) cannot visualize structures smaller than ~1 mm. Microfluidic-scale cilia-driven fluid flow clears pathogen and allergen-containing mucus out of the lungs, yet we currently lack quantitative imaging technologies to characterize their flow performance. Moreover, while ciliary defects are traditionally considered a feature of rare but severe diseases (e.g. primary ciliary dyskinesia), development of biomechanical biomarkers extracted from quantitative flow imaging will allow us to test a paradigm-shifting hypothesis: intermediate defects in ciliary performance that are undetectable by current diagnostics are major modifiers of clinical severity in common respiratory diseases (e.g. asthma). Our research therefore has three aims. First, we will develop high-speed, cosine ambiguity-free Doppler OCT imaging systems. Cilia-driven fluid flow is three-dimensional in nature and not amenable to simplifying geometric assumptions such as parabolic flow profile. Traditional Doppler imaging suffers from cosine ambiguity that precludes the measurement of three-component flow velocities (v=vxi+vyj+vzk). We will develop a novel class of OCT interferometers that will enable three-dimensional, three-component flow imaging that will be demonstrated using the ciliated skin of Xenopus (tadpole) embryos, an important animal model in ciliary biology. Second, we will develop quantitative imaging assays of ciliary function that exploit cilia-driven microfluidic mixing. Taking a cue from work in biomimetic cilia, we have (a) demonstrated that ciliated biological surfaces can drive microfluidic mixing and (b) developed a novel microfluidic chip that uses a ciliated biological surface as a microfluidic "component." Building on these results, we will demonstrate that our microfluidic mixing-based assay can quantify biologically relevant perturbations to ciliary physiology including increased fluid viscosity and altered planar cell polarity. Third, we will demonstrate intermediate defects in Xenopus and mouse ciliary function using quantitative imaging. We will target two different classes of genes relevant in the performance of a ciliated surface in Xenopus embryos: ciliary molecular motors and notch signaling proteins (notch signaling controls the density of ciliated cells on the embryo skin). Given the importance of mouse models of pediatric respiratory disease, it is critical to demonstrate that our optical methods can be used to quantify the performance of mouse respiratory cilia. Moreover, this is an important step towards translating our diagnostic technologies to use in humans. We propose to use flow imaging to quantify performance modified by increased fluid viscosity (mechanical perturbation to decrease ciliary beat frequency) and increased extracellular ATP (pharmacological perturbation to increase ciliary beat frequency).