Novel Approach to Image Mucin Release and Swelling

粘蛋白释放和肿胀成像的新方法

• 批准号: 8721112
• 负责人: ZYGMUNT GRYCZYNSKI
• 金额: $ 17.81万
• 依托单位: TEXAS CHRISTIAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8721112
• 关键词: Acridine Orange; Anisotropy; Area; Asthma; Bacteria; Bicarbonates; Biological Assay; Breathing; Cell physiology; Cells; Chronic Obstructive Airway Disease; Color; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasmic Granules; Detection; Development; Diffusion; Disease; Dyes; Epithelial Cells; Equilibrium; Exhibits; Exocytosis; Film; Fluorescence; Fluorescence Microscopy; Functional Imaging; Gel; Goals; Heterogeneity; Image; Imaging technology; Infection; Investigation; Irritants; Kinetics; Label; Learning; Link; Lung; Membrane; Methods; Microscopy; Monitor; Morbidity - disease rate; Movement; Mucins; Mucociliary Clearance; Mucous body substance; Normal Cell; Optics; Organ; Pathologic; Physiologic pulse; Physiology; Population; Process; Production; Property; Relative (related person); Reporting; Resolution; Rheology; Role; Speed; Surface; Swelling; System; Techniques; Technology; Testing; Thick; Time; Video Microscopy; Virus; Work; absorption; biophysical properties; design; dimer; insight; interest; knock-down; light microscopy; monomer; mortality; new technology; novel; novel strategies; public health relevance; quantum; ratiometric; viscoelasticity

DESCRIPTION (provided by applicant): Mucus secretion is an important mechanism defending from irritants inhaled into the lungs during breathing. Secreted mucus forms a thin film of viscoelastic gel on the surface of the airways that protects the epithelial cells from irriants inhaled into the lungs by entrapping foreign debris, bacteria, and viruses and clearing them from the airway by ciliary movement; the whole process is termed mucociliary clearance. Besides its protective role, mucus could also have a pathologic roles in disease conditions, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD), where excessive production of mucus (hypersecretion) and/or changes in its biophysical properties (viscoelasticity) result in the accumulation of thick, sticky mucus in the lungs, effectively impairing mucociliary clearance process. Since mucus is practically colorless viscous substance current video and light microscopy techniques used for studying mucus secretion suffer from poor resolution, sensitivity and limited temporal resolution. Fluorescence studies of mucin secretion are hampered by lack of well characterized fluorescent labels of mucin molecules and by difficulty to image in real-time a very rapid (~100 ms) secretion and swelling process. We recently tried a fluorescence approach with various fluorescence dyes to enable monitoring of mucin release on the cellular level and realized that the most interesting (important) processes occur in the initial steps when granules and generated mucus patches are small, frequently bellow optical resolution limits (<0.5 ?m). In this situation it is impossible to quantitatively reate fluorescence intensity changes (decreases) to mucus swelling. But one probe we tested (Acridine Orange - AO) exhibited very promising properties. The probe very effectively accumulates in low pH mucus granules inside the cell forming dimers/aggregates. The emission of the dimer/aggregate form is shifted 100 nm toward the red and its fluorescence lifetime is very long (over 10 times longer than fluorescence lifetime of the monomer). Also the brightness of aggregates is relatively high, making their detection easier. When mucus is released and swells, the equilibrium between monomers and aggregates quickly shifts toward monomers, producing a distinct change in color (from red to green). We expect this could be a great opportunity to develop/utilize the first fluorescence probe for studying the kinetics of mucus expansion. AO is one of the longest known fluorescent markers, and it was a surprise for us to realize that the amount of the information regarding the monomer-dimer/aggregates equilibrium is very limited. Multiple studies done over the past 50 years only give limited and partial information because of technical difficulties that were impossible to overcome with the available technologies at the time. We conducted initial studies of AO properties and immediately realized that this probe will have great potential for studying exocytotic processes. In this application we propose to use what we learned from our preliminary work to develop the application of AO in investigation of the kinetics of the mucus formation process. Our goal is to establish new technology for imaging cellular processes associated with mucus release with high spatial and temporal resolution. Distinctly different spectroscopic properties of the monomeric and aggregated form of AO open a novel possibility for the development of a two excitation wavelength, ratiometric, TIRF- FLIM approach that will allow, for the first time, detailed kinetics studies of mucin swelling and monitoring of its rheologic (viscoelastic) properties at the single mucin granule level. Total internal reflection fluorescence (TIRF) will allow surface confined excitation for monitoring membrane processes within a 100 nm layer. Fluorescence lifetime imaging (FLIM) will allow very precise, fast detection on the monomer-aggregate equilibrium independently of the granule size. Use of two excitation wavelengths as interleaved pulses with adjustable relative delay will allow simultaneous monitoring of monomer and aggregate populations thus highly increasing sensitivity and speed for detection.

描述（由申请人提供）：粘液分泌是捍卫刺激物在呼吸过程中吸入肺部的重要机制。分泌的粘液在气道表面形成了薄膜的薄膜，通过夹住外国碎屑，细菌和病毒来保护上皮细胞免受液体吸入肺部的炎症，并通过睫状运动清除它们免受气道的清除；整个过程称为粘毛清除率。除保护作用外，粘液还可以在疾病中具有病理作用，例如囊性纤维化（CF），哮喘和慢性阻塞性肺部疾病（COPD），其中粘液过多的粘液产生（过度分泌）和/或其生物物质变化性质（粘弹性）导致肺部厚实粘性的积累，从而有效地损害了粘膜纤毛清除过程。由于粘液实际上是无色粘性物质的当前视频和光学显微镜技术，用于研究粘液分泌的分辨率差，敏感性和时间分辨率有限。粘蛋白分泌的荧光研究由于缺乏粘蛋白分子的荧光标签而受到阻碍，并且难以实时成像非常快速（〜100 ms）的分泌和肿胀过程。最近，我们尝试了一种具有各种荧光染料的荧光方法，以在细胞水平上监测粘蛋白释放，并意识到，当颗粒和产生的粘液斑块是小的，通常是波纹的光学分辨率限制时，最有趣的（重要）过程发生在初始步骤中（ <0.5？m）。在这种情况下，不可能定量重新重新荧光强度变化（降低）以粘液肿胀。但是我们测试的一项探针（Acridine Orange -AO）表现出非常有希望的特性。探针非常有效地积聚在细胞形成二聚体/聚集体内部的低pH粘液颗粒中。二聚体/聚集体形式的发射向红色转移100 nm，其荧光寿命非常长（比单体的荧光寿命长10倍）。同样，聚集体的亮度相对较高，从而使它们的检测更加容易。当粘液释放并膨胀时，单体和聚集体之间的平衡迅速向单体转移，从而产生颜色的明显变化（从红色到绿色）。我们预计这可能是一个很好的机会，可以开发/利用第一个荧光探针来研究粘液膨胀动力学。 AO是已知最长的荧光标记之一，令我们意识到有关单体二聚体/聚集体平衡的信息的量非常有限。在过去的50年中进行的多项研究仅提供有限的部分信息，因为当时的可用技术无法克服的技术困难。我们对AO特性进行了初步研究，并立即意识到该探针将具有研究胞吐过程的巨大潜力。在此应用中，我们 建议利用我们从初步工作中学到的知识来开发AO在调查粘液形成过程动力学中的应用。我们的目标是建立与粘液释放相关的细胞过程的新技术，并具有高空间和时间分辨率。 AO的单体和聚合形式的光谱特性截然不同 对单个粘蛋白颗粒水平的流变学（粘弹性）特性的粘蛋白肿胀和监测的研究。总内反射荧光（TIRF）将允许表面限制激发，以监测100 nm层内的膜过程。荧光寿命成像（FLIM）将允许与颗粒大小独立于单体 - 聚集酸盐平衡进行非常精确的快速检测。将两个激发波长用作具有可调相对延迟的交织脉冲，将允许同时监测单体和聚集体的群体，从而高度提高灵敏度和检测速度。