Quantitative optical imaging of cilia-driven fluid flow

描述（由申请人提供）：呼吸道疾病是儿童发病和死亡的主要原因。人们对这些疾病还不完全了解，这是改善临床护理的一个障碍。因此，需要发现新的疾病机制。光学成像（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（如x射线、CT、MRI）无法显示小于1毫米的结构。微流体规模的纤毛驱动的流体流动清除了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疾病（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验证一个范式转移的假设：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（如哮喘）临床严重程度的主要改变因素。因此，我们的研究有三个目的。首先，我们将开发高速，余弦无模糊的多普勒OCT成像系统。纤毛驱动的流体流动本质上是三维的，不能简化几何假设，如抛物线流剖面。传统的多普勒成像存在余弦模糊，无法测量三分量流速（v=vxi+vyj+vzk）。我们将开发一种新型的OCT干涉仪，它将实现三维，三组分流成像，将使用爪蟾（蝌蚪）胚胎的纤毛皮肤进行演示，这是纤毛生物学中重要的动物模型。其次，我们将开发利用纤毛驱动微流体的纤毛功能定量成像分析

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).