Novel Approach to Image Mucin Release and Swelling

描述(申请人提供)：粘液分泌是防止刺激物在呼吸过程中吸入肺部的重要机制。分泌的粘液在呼吸道表面形成一层粘弹性凝胶薄膜，通过捕获外来碎片、细菌和病毒并通过纤毛运动将它们从呼吸道清除，从而保护上皮细胞免受吸入肺部的刺激物的伤害；整个过程被称为粘液纤毛清除。除了保护作用外，粘液还可以在囊性纤维化(CF)、哮喘和慢性阻塞性肺疾病(COPD)等疾病中发挥病理作用，在这些疾病中，粘液的过度产生(高分泌)和/或其生物物理特性(粘弹性)的变化会导致粘液在肺部积聚，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，目前用于研究粘液分泌的视频和光学显微镜技术分辨率低、灵敏度低、时间分辨率有限。由于缺乏表征良好的粘蛋白分子的荧光标记，以及难以实时成像非常快速(~100ms)的分泌和肿胀过程，阻碍了对粘蛋白分泌的荧光研究。我们最近尝试了一种使用各种荧光染料的荧光方法，以在细胞水平上监控粘蛋白的释放，并意识到最有趣(重要)的过程发生在最初的步骤中，此时颗粒和产生的粘液斑块很小，通常低于光学分辨率极限(&lt；0.5？m)。在这种情况下，不可能将荧光强度的变化(减弱)定量地表示为粘液肿胀。但我们测试的一种探针(Acridine Orange-AO)显示出非常有希望的特性。该探针非常有效地聚集在细胞内的低pH粘液颗粒中，形成二聚体/聚集体。二聚体/聚集体的发射向红移了100 nm，荧光寿命很长(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，更容易检测到它们。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的动力学。AO是已知最长的荧光标记之一，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不可能克服这些技术困难。我们对氧的性质进行了初步研究，并立即意识到该探针将在研究胞吐过程中具有巨大的潜力。在此应用程序中，我们 建议利用我们从前期工作中所学到的知识来发展AO在粘液形成过程的动力学研究中的应用。我们的目标是建立与粘液释放相关的高空间和时间分辨率的细胞过程成像的新技术。单体和聚集态的明显不同的光谱性质为开发双激发波长、比率、TIRF-Flim方法打开了新的可能性，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膨胀及其流变性(粘弹性)的监测。全内反射荧光(TIRF)将允许表面受限激发来监测100 nm层内的膜过程。荧光寿命成像(FLIM)将允许非常精确、快速地检测与颗粒大小无关的单体-聚集体平衡。使用两个激发波长作为相对延迟可调的交错脉冲，将允许同时监测单体和聚集体，从而极大地提高检测的灵敏度和速度。

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.