Quantitative optical imaging of cilia-driven fluid flow

描述(申请人提供)：呼吸系统疾病是儿科发病率和死亡率的主要原因。对这些疾病的了解还不完全，这是改善临床护理的障碍。因此，需要发现新的致病机制。光学成像(例如光学相干层析成像[OCT])将使这些发现成为可能，因为传统成像(例如X射线、CT、MRI)无法可视化小于1 mm的结构。微流控纤毛驱动的液体流动可以清除肺部的病原体和含有过敏原的粘液，但我们目前缺乏定量成像技术来表征它们的流动性能。此外，虽然纤毛缺陷传统上被认为是罕见但严重的疾病(如原发性睫状体运动障碍)的特征，但从定量血流成像提取的生物力学生物标记物的发展将使我们能够检验一个范式转换假说：目前诊断无法发现的纤毛性能中等缺陷是常见呼吸系统疾病(如哮喘)临床严重程度的主要修饰因素。因此，我们的研究有三个目标。首先，我们将开发高速、无余弦模糊的多普勒OCT成像系统。纤毛驱动的流体流动本质上是三维的，不服从简化的几何假设，如抛物线流型。传统的多普勒成像存在余弦模糊问题，无法测量三分量血流速度(v=vxi、vyj、vzk)。我们将开发一种新型的OCT干涉仪，它将实现三维三组分流动成像，并将使用非洲爪哇(蝌蚪)胚胎的纤毛皮肤进行演示，这是纤毛生物学中的一个重要动物模型。其次，我们将利用纤毛驱动的微流控技术开发睫毛功能的定量成像分析。 混合。我们从仿生纤毛的工作中得到了启示，(A)证明了纤毛生物表面可以驱动微流控混合，(B)开发了一种新型的微流控芯片，它使用纤毛生物表面作为微流控“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检测方法可以量化纤毛生理的生物相关扰动，包括流体粘度增加和平面细胞极性改变。第三，我们将使用定量成像来展示非洲爪哇和小鼠睫毛功能的中度缺陷。我们将针对两类与非洲爪哇胚胎纤毛表面性能相关的基因：纤毛分子马达和Noch信号蛋白(Noch信号控制胚胎皮肤上纤毛细胞的密度)。鉴于儿科呼吸道疾病小鼠模型的重要性，证明我们的光学方法可以用于量化小鼠呼吸道纤毛的表现是至关重要的。此外，这是将我们的诊断技术转化为用于人类的重要一步。我们建议使用血流成像来量化由于增加液体粘度(机械扰动以降低纤毛搏动频率)和增加细胞外ATP(药物扰动以增加纤毛搏动频率)而修改的性能。

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).