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）：粘液分泌是呼吸期间抵抗吸入肺部的刺激物的重要机制。分泌的粘液在气道表面上形成粘弹性凝胶薄膜，其通过截留外来碎片、细菌和病毒并通过纤毛运动将它们从气道清除来保护上皮细胞免受吸入肺中的冲洗剂的影响;整个过程被称为粘液纤毛清除。除了其保护作用之外，粘液还可以在疾病状况中具有病理作用，例如囊性纤维化（CF）、哮喘和慢性阻塞性肺病（COPD），其中粘液的过度产生（分泌过多）和/或其生物物理性质（粘弹性）的变化导致肺中粘稠粘液的积累，从而有效地损害粘液纤毛清除过程。由于粘液实际上是无色粘性物质，因此用于研究粘液分泌的当前视频和光学显微镜技术遭受分辨率差、灵敏度和有限的时间分辨率。粘蛋白分泌的荧光研究由于缺乏粘蛋白分子的良好表征的荧光标记以及难以实时成像非常快速（~100 ms）的分泌和膨胀过程而受到阻碍。我们最近尝试了一种荧光方法与各种荧光染料，使监测粘蛋白释放的细胞水平上，并意识到，最有趣的（重要的）过程发生在最初的步骤时，颗粒和产生的粘液补丁是小的，经常低于光学分辨率限制（<0.5？m）。在这种情况下，不可能定量地将荧光强度变化（降低）与粘液肿胀相关联。但我们测试的一种探针（吖啶橙子- AO）表现出非常有前途的特性。探针非常有效地积聚在细胞内的低pH粘液颗粒中，形成二聚体/聚集体。二聚体/聚集体形式的发射向红色移动100 nm，并且其荧光寿命非常长（比单体的荧光寿命长10倍以上）。此外，聚集体的亮度相对较高，使其更容易检测。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，产生明显的颜色变化（从红色到绿色）。我们希望这可能是一个很好的机会，开发/利用第一个荧光探针研究粘液扩张的动力学。AO是已知最长的荧光标记物之一，并且令我们惊讶的是，关于单体-二聚体/聚集体平衡的信息量非常有限。过去50年来进行的多项研究只提供了有限和部分的信息，因为当时的现有技术无法克服技术困难。我们进行了初步的研究AO性能，并立即意识到，这种探针将有很大的潜力，研究胞吐过程。在本申请中，我们 建议使用我们从我们的初步工作中学到的知识来开发AO在粘液形成过程的动力学研究中的应用。我们的目标是建立一种新的技术，以高的空间和时间分辨率成像与粘液释放相关的细胞过程。AO的单体和聚集形式的明显不同的光谱性质为开发双激发波长、比率计量、TIRF-FLIM方法开辟了新的可能性，该方法将首次允许详细的动力学 在单个粘蛋白颗粒水平上研究粘蛋白溶胀和监测其流变学（粘弹性）性质。全内反射荧光（TIRF）将允许表面限制激发用于监测100 nm层内的膜过程。荧光寿命成像（FLIM）将允许非常精确，快速检测的单体聚集体的平衡独立的颗粒大小。使用两个激发波长作为具有可调相对延迟的交错脉冲将允许同时监测单体和聚集体群体，从而高度增加检测的灵敏度和速度。